Recombinant vaccine against West Nile Virus

ABSTRACT

An immunogenic or vaccine composition to induce an immune response or protective immune response against West Nile virus (WNV) in an animal susceptible to WNV. The composition includes a pharmaceutically or veterinarily acceptable vehicle or excipient, and a vector. The vector contains heterologous nucleic acid molecule(s), expresses in vivo in the animal WNV antigen, immunogen or epitope thereof, e.g., WNV E; WNV prM and E; WNV M and E; WNV prM, WNV M and E, WNV polyprotein prM-E, WNV polyprotein M-E, or WNV polyprotein prM-M-E. The composition can contain an adjuvant, such as carbomer. Methods for making and using such a composition, including prime-boost regimes and including as to differential diagnosis, are also contemplated.

RELATED APPLICATIONS/INCORPORATION BY REFERENCE

This application is a continuation of U.S. application Ser. No.10/714,781 filed on Nov. 17, 2003, which is a continuation-in-partapplication of U.S. application Ser. No. 10/679,520 filed Oct. 6, 2003,which is a continuation-in-part application of U.S. application Ser. No.10/374,953 filed Feb. 26, 2003, which is a continuation-in-part of U.S.application Ser. No. 10/116,298, filed Apr. 4, 2002, which claimspriority from U.S. Provisional application Ser. No. 60/281,923, filedApr. 6, 2001. Application Ser. No. 10/714,781 filed on Nov. 17, 2003, isalso a continuation-in-part application of U.S. application Ser. No.10/676502, filed Sep. 30, 2003, which is a continuation of U.S.application Ser. No. 10/374,953 filed Feb. 26, 2003. Each of the aboveapplications, together with each document cited therein, and each of thedocuments referenced or cited in documents cited therein, are herebyincorporated herein by reference.

Indeed, more generally, each document cited in this text (“applicationcited documents”) and each document cited or referenced in each of theapplication cited documents, and any manufacturer's specifications orinstructions for any products mentioned in this text and in any documentincorporated into this text, are hereby incorporated herein byreference; and, technology in each of the documents incorporated hereinby reference can be used in the practice of this invention.

FIELD OF THE INVENTION

The present invention relates to vectors containing at least onepolynucleotide of the West Nile fever virus (or West Nile Virus or WNV)or at least one nucleic acid molecule encoding at least one West NileVirus antigen, immunogen or epitope, e.g., in vivo and in vitroexpression vectors comprising and expressing at least one polynucleotideof the West Nile Virus or in vivo and in vitro expression vectorscomprising and expressing at least one West Nile Virus antigen,immunogen or epitope, as well as immunogenic compositions and vaccinesagainst West Nile fever; for instance, such compositions or vaccinesthat contain one or more of the vectors and/or one or more of theexpression products of the vectors. The invention also relates tomethods for using the vectors, compositions and vaccines, including forimmunizing and vaccinating against this virus, expressing expressionproducts of the polynucleotide(s), using the expression products inassays or to generate antibodies useful in assays, as well as to methodsfor making the, polynucleotide(s), vectors, compositions vaccines,assays, inter alia.

BACKGROUND OF THE INVENTION

The West Nile fever virus (WNV) was first identified in man in 1937 inUganda in the West Nile Province (Zeller H. G., Med. Trop., 1999, 59,490-494).

Widespread in Africa, it is also found in India, Pakistan and theMediterranean basin and was identified for the first time in the USA in1999 in New York City (Anderson J. F. et al., Science, 1999, 286,2331-2333).

The West Nile fever virus affects birds as well as reptiles, mammals,together with man.

The disease is characterized in birds by an attack of the centralnervous system and death. The lesions include encephalitis, hemorrhagesin the myocardium and hemorrhages and necroses in the intestinal tract.

In chickens, experimental infections by subcutaneous inoculations of theWest Nile fever virus isolated on crows led to necrosis of themyocardium, nephritis and pneumonia 5 to 10 days after inoculation andmoderate to severe encephalitis 21 days after inoculation (Senne D. A.et al., Avian Disease, 2000, 44, 642-649).

The West Nile fever virus also affects horses, especially in NorthAfrica and Europe (Cantile C. et al., Equine Vet. J., 2000, 32 (1),31-35). These horses reveal signs of ataxia, weakness of the rear limbs,paresis evolving towards tetraplegia and death. Horses and camels arethe main animals manifesting clinical signs in the form of encephalitis.

Anti-WNV antibodies were detected in certain rodents, in livestock,especially bovines and ovines, as well as in domestic animals,especially in the dog (Zeller H. G., Med. Trop., 1999, 59, 490-494;Lundstrom J. O., Journal of Vector Ecology, 1999, 24 (1), 1-39).

The West Nile fever virus also affects with a number of symptoms thehuman species (Sampson B. A., Human Pathology, 2000, 31 (5), 527-531;Marra C. M., Seminars in Neurology, 2000, 20 (3), 323-327).

The West Nile fever virus is transmitted to birds and mammals by thebites of certain mosquitoes (e.g. Culex, Aedes, Anopheles). Directtransmission may happen from WNV infected subject to healthy subject byoral transmission (prey and transmission through colostrum) andblood/organ vectored transmission.

Wild and domestic birds are a reservoir for the West Nile virus and apropagation vector as a result of their migrations.

The virions of the West Nile fever virus are spherical particles with adiameter of 50 nm constituted by a lipoproteic envelope surrounding anicosahedric nucleocapsid containing a positive polarity, single-strandRNA.

A single open reading frame (ORF) encodes all the viral proteins in theform of a polyprotein. The cleaving and maturation of this polyproteinleads to the production of about ten different viral proteins. Thestructural proteins are encoded by the 5′ part of the genome andcorrespond to the nucleocapsid designated C (14 kDa), the envelopeglycoprotein designated E (50 kDa), the pre-membrane protein designatedprM (23 kDa), the membrane protein designated M (7 kDa). Thenon-structural proteins are encoded by the 3′ part of the genome andcorrespond to the proteins NS1 (40 kDa), NS2A (19 kDa), NS2B (14 kDa),NS3 (74 kDa), NS4A (15 kDa), NS4B (29 kDa), NS5 (97 kDa).

Parrish C. R. et al. (J. Gen. Virol., 1991, 72, 1645-1653), Kulkarni A.B. et al. (J. Virol., 1992, 66 (6), 3583-3592) and Hill A. B. et al. (J.Gen. Virol., 1992, 73, 1115-1123), on the basis of the vaccinia virus,constructed in vivo expression vectors containing various insertscorresponding to nucleotide sequences coding for non-structural proteinsof the Kunjin virus, optionally associated with structural proteins.These vectors were administered to mice to evaluate the immune cellresponse. The authors stress the importance of the cell response, whichis essentially stimulated by non-structural proteins and especially NS3,NS4A and NS4B. These articles reveal the difficulty in providing a goodvaccination strategy against West Nile fever.

Reference is also made to WO 02/081754 published Oct. 17, 2002, fromPCT/US02/10764, filed Apr. 4, 2002, with a claim of priority from U.S.application Ser. No. 09/826,115, filed Apr. 4, 2001. The PCT claims astatus of continuation-in-part from U.S. application Ser. No.09/826,115. It further states that U.S. application Ser. No. 09/826,115is a continuation-in-part of U.S. application Ser. No. 09/701,536, filedNov. 29, 2000. It even further states that U.S. application Ser. No.09/701,536 is the National Stage of PCT/US99/12298, filed Jun. 3, 1999,with a claim of priority to U.S. provisional application Ser. No.60/087,908.

It would be advantageous to provide improved immunogenic and vaccinecompositions against WNV, and methods for making and using suchcompositions, including such compositions that provide for differentialdiagnostic methods, assays and kits, and thus, differential diagnosticmethods, assays and kits.

OBJECTS AND/OR SUMMARY OF THE INVENTION

The invention provides an immunogenic or vaccine composition to inducean immune response or protective immune response against West Nile virus(WNV) in an animal susceptible to WNV comprising or consistingessentially of a pharmaceutically or veterinarily acceptable vehicle orexcipient and a vector that contains or consists essentially ofheterologous nucleic acid molecule(s), and that expresses in vivo in theanimal a WNV protein, antigen, immunogen or epitope thereof, such as WNVE; WNV prM and E; WNV M and E; WNV prM, WNV M and E, WNV polyproteinprM-E, WNV polyprotein M-E, or WNV polyprotein prM-M-E.

The vector can be a DNA plasmid or a recombinant virus, such as arecombinant adenovirus, herpesvirus or poxvirus, e.g., an avipox virus,such as a canarypox virus or a fowlpox virus. The animal can be selectedfrom the group consisting of an equine, a canine, a feline, a bovine, aporcine, a chicken, a duck, a goose and a turkey.

Advantageously, the nucleic acid molecule comprises or consistsessentially of nucleotides 466-741, 742-966 and 967-2469 of GenBankAF196835 encoding WNV prM, M and E, respectively, nucleotides 466-2469of GenBank AF196835 encoding WN protein prM-M-E, or nucleotides 421-2469of GenBank AF196835 encoding WN protein prM-M-E and the signal peptideof prM.

The immunogenic or vaccine composition can further comprise or consistessentially of an adjuvant, such as a carbomer.

The immunogenic or vaccine composition can further comprise or consistessentially of an antigen or immunogen or epitope thereof of a pathogenother than WNV of the animal, or a vector that contains and expresses invivo in the animal a nucleic acid molecule encoding the antigen,immunogen or epitope thereof, or an inactivated or attenuated pathogenother than WNV of the animal.

The invention additionally involves a kit comprising or consistingessentially of (a) the immunogenic or vaccine composition, and (b) theantigen or immunogen or epitope thereof of a pathogen other than WNV ofthe animal, or the vector that contains and expresses in vivo in theanimal a nucleic acid molecule encoding the antigen, immunogen orepitope thereof, or the inactivated or attenuated pathogen other thanWNV of the animal, wherein (a) and (b) are in separate containers, andthe kit optionally contains instructions for admixture and/oradministration of (a) and (b).

The invention also comprehends a method for inducing an immunological orprotective immune response against WNV in an animal comprising orconsisting essentially of administering to the animal the immunogenic orvaccine composition.

The invention further comprehends a method for inducing an immunologicalor protective immune response against WNV in an animal comprising orconsisting essentially of administering to the animal (a) theimmunogenic or vaccine composition, and (b) a WNV isolated antigen,immunogen or epitope thereof, wherein (a) is administered prior to (b)in a prime-boost regimen, or (b) is administered prior to (a) in aprime-boost regimen, or (a) and (b) are administered together, eithersequentially or in admixture. The invention also involves a kit forperforming this comprising or consisting essentially of (a) and (b) inseparate containers, optionally with instructions for admixture and/oradministration.

The invention even further comprehends a prime-boost immunization orvaccination against WNV, wherein the priming is done with (a) DNAvaccine(s) or immunological or immunogenic composition(s) that containsor consists essentially of (a) nucleic acid molecule(s) encoding andexpress(es) in vivo a WNV immunogen, antigen or epitope and the boost isdone with (a) vaccine(s) or immunological or immunogenic composition(s)that is a WNV inactivated or attenuated or subunit (antigen, immunogenand/or epitope) preparation(s) and/or (a) recombinant or modified virusvaccine or immunological or immunogenic composition(s) that contains orconsists essentially of (a) nucleic acid molecule encoding andexpress(es) in vivo (a) WNV immunogen(s), antigen(s) or epitope(s).Thus, the invention provides a prime-boost immunization or vaccinationmethod against WNV, such as a prime-boost immunization or vaccinationwhich comprises or consists essentially of or consists of administeringto a target species animal (a) DNA vaccine(s) or immunological orimmunogenic composition(s) of the invention (that contains or consistsessentially of nucleic acid molecule(s) encoding and express(es) in vivoWNV antigen(s), immunogen(s) or epitope(s)) (as the prime) andthereafter administering (as the boost) administering inactivated WNVand/or attenuated WNV or a WNV subunit (antigen, immunogen and/orepitope) preparation(s)) and/or a recombinant or modified virus vaccineor immunological or immunogenic composition that contains or consistsessentially of nucleic acid molecule(s) encoding and expresse(s) in vivoWNV immunogen(s), antigen(s) or epitope(s), advantageously (a)recombinant vaccine or immunological or immunogenic composition(s) thatexpresses the WNV immunogen, antigen or epitope in vivo. The boost isadvantageously matched to the prime, e.g., the boost contains orconsists essentially of or expresses at least one antigen, epitope orimmunogen that is expressed by the prime.

The prime-boost regimen according to the invention can be used inanimals of any age, advantageously young animals (e.g., animals thathave detectable maternal antibodies and/or are suckling or nursing orbreast-feeding), pre-adult animals (animals that are older than being ayoung animal but have not yet reached maturity or adulthood or an age tomate or reproduce), adult animals (e.g., animals that are of an age tomate or reproduce or are beyond such a period in life), and it isadvantageous to employ the prime-boost regimen in pregnant females orfemales prior to giving birth, laying, or insemination.

The invention also relates to such immunogenic and vaccine compositionsand kits thereof suitable for use in such prime-boost regimens andprime-boost regimens. The host or target species upon which theprime-boost regimen can be practiced includes any animal (target orhost) species susceptible to disease caused by WNV, including mammals,reptiles, birds, especially humans, companion mammals or animals such ascanines, felines, equines, zoo mammals or animals, such as aquaticmammals e.g. seals, felines, equines, zoo reptiles such as snakes,crocodiles, aligators, and avian species, such as domesticated birdsthat are pets or poultry, or wild birds, e.g., canaries, parakeets,chickens, ducks, geese, turkeys, sparrows, crows, and the like.

The prime-boost regimen is especially advantageous to practice in ayoung animal, as it allows vaccination or immunization at an early age,for instance, the first administration in the prime-boost regimen whenpracticed on a young animal can be at an age at which the young animalhas maternal antibodies. Another advantage of this regimen is that itcan provide a degree of safety for pregnant females present in the samelocation or in close proximity to the young or to each other. Thus, theinvention provides a prime-boost immunization or vaccination methodagainst WNV, and the method may be practiced upon a young animal, suchas a young foal, puppy or kitten, for instance, wherein the priming isdone at a time that the young animal has maternal antibodies againstWNV, with the boost advantageously at a time when maternal antibodiesmay be waning or decreasing or normally not present, such as a period oftime post-breastfeeding.

Accordingly, the invention also involves kits for performing aprime-boost regimen comprising or consisting essentially of a primingvaccine or immunological or immunogenic composition and a boost vaccineor immunological or immunogenic compositions, in separate containers,optionally with instructions for admixture and/or administration.

Further still, the invention provides a differential diagnosis methodcomprising administering to animals an immunogenic or vaccinecomposition and/or a WNV antigen, immunogen or epitope, and testing theanimals for presence or absence of a WNV protein or antibody thereto notexpressed by the immunogenic or vaccine composition and/or not presentin the WNV antigen, immunogen or epitope. An the invention additionallyinvolves a kit for performing this method comprising the immunogenic orvaccine composition and/or the WNV antigen, immunogen or epitope, and anassay for testing for the presence or absence of the WNV protein, inseparate containers, optionally with instructions for administration ofthe immunogenic or vaccine composition and/or the WNV antigen, immunogenor epitope and/or for performing the assay.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing construction of a pC5 H6p WNV prM-M-Edonor plasmid, pDS-2946-1-1.

FIG. 2 depicts the nucleic and amino acid sequence of C5 H6p WNV prM-M-EC5 in pDS-2646-1-1 (SEQ ID Nos: 54 and 55).

FIG. 3 is a schematic showing construction of a pF8 H6p WNV prM-M-Edonor plasmid, pSL-5513-1-1-1.

FIG. 4 depicts the nucleic and amino acid sequence of F8 H6p WNV prM-M-EF8 in pSL-5513-1-1-1 (SEQ ID Nos: 56 and 57).

FIG. 5 is two immunoblots showing the expression of WNV proteins frompox recombinants in chick embryo fibroblast cells.

FIG. 6 is an immunoblot showing the expression of WNV proteins from poxrecombinants in BHK cells.

FIG. 7 is a schematic showing construction of pVR1012WNV prM-M-E,pSL-5448-1-1.

FIG. 8 depicts the nucleic and amino acid sequences of the WNV prM-M-Eregion in pSL-5448-1-1, pVR1012WNV prM-M-E (SEQ ID Nos: 58 and 59).

FIG. 9 depicts the nucleic and amino acid sequences of pDS-2946-1-1, pC5H6p WNV prM-M-E (SEQ ID Nos: 60 and 61).

FIG. 10 is a schematic showing construction of pC5 H6p WNV prM-M-E donorplasmids with a truncated H6p and/or truncated WNV capsid leadersequence.

FIG. 11 describes primers for constructions pC5 H6p WNV prM-M-E donorplasmids with a truncated H6p and/or truncated WNV capsid leadersequence (SEQ ID NOS: 63, 62, 50, 49, 48, 65, and 64, respectively inorder of appearance). Additionally, a fragment of the full lengthsequence of H6p 5′WNV sequence in vCP2017 is depicted (as bases1861-2160 of SEQ ID NO: 60 and residues 1-52 of SEQ ID NO:61).

FIG. 12 depicts the nucleic and amino acid sequences of the West NileVirus (WNV) (SEQ ID Nos: 66 and 67).

FIG. 13 (SEQ ID NO:77) is the sequence of a 5 kb segment of canarypoxDNA. encoding an ORF designated C5 initiating at position 1864 andterminating at position 2187 (SEQ ID NO:78). The oligonucleotides shownare disclosed as SEQ ID NOS:79-81, respectively in order of appearance.

FIG. 14 depicts the sequence of a 232 by VQ/H6p/MCS fragment. Thenucleotide and amino acid sequences are disclosed as SEQ ID NOS:82 and83, respectively.

FIG. 15 is a schematic showing the generation of plasmid pNVQH6C5LSP-18,a C5 insertion plasmid containing the H6 promoter, transcription andtranslation terminators functional in all reading frames, and a MCS.

FIG. 16 is a table that provides data depicting the plaque-forming unitsof WNV per ml of serum for each of the horses in Example 32

FIG. 17 a-b is a table that provides data showing the presence orabsence of virenia based on a serum titter of titer of > or <5 pfu/ml[log₁₀<0.7].

FIG. 18 a-b is a table providing temperature data for each of the horsesin Example 32.

FIG. 18 c is a graph depicting the temperature data as an average foreach of Groups I and II.

FIG. 19 a-b is a table providing data from plaque reductionneutralization titers in all of the animals in groups I and II.

DETAILED DESCRIPTION

As discussed herein, the present invention relates to vectors containingat least one polynucleotide of the West Nile fever virus (or West NileVirus or WNV) or at least one nucleic acid molecule encoding at leastone West Nile Virus antigen, immunogen or epitope, e.g., in vivo and invitro expression vectors comprising and expressing at least onepolynucleotide of the West Nile Virus or in vivo and in vitro expressionvectors comprising and expressing at least one West Nile Virus antigen,immunogen or epitope, as well as immunogenic compositions and vaccinesagainst West Nile fever; for instance, such compositions or vaccinesthat contain one or more of the vectors and/or one or more of theexpression products of the vectors.

Advantageously, the immunogen or antigen is the envelope protein, E, orthe pre-membrane protein (prM protein), or the membrane protein (Mprotein), or combinations thereof, e.g., E and prM; E and M; E and prMand M; prM and M. The combinations can be separate proteins orpolyproteins. The compositions or vaccines can thus contain one or morevectors expressing more than one of the proteins, e.g., differentproteins. The compositions or vaccines can contain, or vectors thereofexpress, proteins from different strains or isolates of WNV. Thus, thecompositions or vaccines can contain, or the vectors thereof express, E,prM, M or combinations thereof, wherein the E, prM, and/or M are fromdifferent strains or isolates.

In this regard, it is noted that there is the NYC isolate or strain,e.g., WN-NY99 strain or GenBank AF196835 (WNV isolated from a deadChilean flamingo at the Bronx Zoo deposited in GenBank, R. S. Lanciottiet al., Science, 286, pp. 2333-7 (1999); SEQ ID Nos: 66 and 67) orGenBank AAF202541 (genome of a WNV isolate from human victims of the NewYork outbreak of WNV-NY1999, X-Y. Jia et al., The Lancet, 354, pp.1971-2 (1999)) (see also Ebel et al., Emerg Infect Dis 7(4):650-3(2001), Anderson et al. PNAS USA 98(23):12885-9 (2001), Shi et al.,Virology 296(2):219-33 (2002), Shi et al., J Virol 76(12):5847-56(2002)), as well as the strains of GenBank D00246 (Kunjin virus); M12294(West Nile virus); AF130362 (West Nile virus strain RO97-50); AF130363(West Nile virus strain 96-1030)). Also, it is noted that comparativephylogenetic analysis of the NY sequences with previously reported WNVsequences indicated a high degree of homology between the NY isolatesand two isolates from Romania and one from Israel (J. F. Anderson etal., supra; X.-Y. Jia et al., supra; R. S. Lanciotti et al., supra),indicating the useful of the NY sequences.

Advantageously in embodiments involving at least one epitope present in,or expressed by vector or vectors in, compositions or vaccines of theinvention, the epitope or epitopes are from E, prM, M or combinationsthereof, and the epitope or epitopes can be from different strains orisolates. In this regard, it is noted that one can locate or mapepitopes in WNV antigens or immunogens, such as the E protein; see,e.g., Beasley et al. J Virol 76(24):13097-100 (2002), Damle et al. ActaVirol 42(6):389-95 (1998), De Groot et al., Emerg Infect Dis 7(4):706-13(2001), Sbai et al., Curr Drug Targets Infect Disord 1(3):303-13 (2001),Kutubuddin et al., Mol Immunol 28(1-2):149-54 (1991), Becker, VirusGenes 4(3):267-82 (1990).

Also as discussed herein, the invention relates to methods for using thevectors, compositions and vaccines, including for immunizing andvaccinating against this virus, for expressing expression products ofthe polynucleotide(s), and methods for using the expression products inassays or to generate antibodies useful in assays, as well as to methodsfor making the, polynucleotide(s), vectors, compositions vaccines,assays, inter alia.

The present invention thus relates to means for preventing and/orcombating diseases caused by the WNV.

The invention relates to such immunogenic and vaccine compositionssuitable for use in different animal (target or host) speciessusceptible to disease caused by WNV, including mammals, reptiles,birds, especially humans, companion mammals or animals such as canines,felines, equines, zoo mammals or animals, such as aquatic mammals e.g.seals, felines, equines, zoo reptiles such as snakes, crocodiles,aligators, and avian species, such as domesticated birds that are petsor poultry, or wild birds, e.g., canaries, parakeets, chickens, ducks,geese, turkeys, sparrows, crows, and the like.

The invention further relates to immunization and vaccination methodsinvolving the immunogenic and vaccine compositions, for the target orhost species. And on this aspect of the invention, mention is made thatas to wild or non-domesticated animals, such as wild or non-domesticatedbirds or mammals (e.g., raccoons, squirrels, mice, or more generallyrodents, felines, canines, etc.) compositions comprising one or morevectors that express one or more WNV epitopes or antigens or immunogenscan be delivered via food, e.g., a bait drop, or mammal or bird food,left for consumption by wild or non-domesticated birds or mammals, thatincludes or contains the one or more vectors, so there may beadministration thereof orally by the mammal or bird consuming the food.This route of administration may be advantageous when the one or morevectors is one or more poxviruses, e.g., an avipox virus such as anattenuated canarypox virus, for instance ALVAC, or an attenuated fowlpoxvirus, for instance TROVAC, or a vaccinia virus, such as an attenuatedvaccinia virus, for instance NYVAC. Accordingly, the invention envisionsoral or mucosal administration, as well as edible compositions thatcontain one or more of the inventive vectors, akin to the MERIAL rabiesproduct RABORAL. From this disclosure and the knowledge in the art, theskilled artisan can formulate edible animal feed for a bird or mammalthat contains a suitable dose of one or more inventive vectors.Furthermore, the invention comprehends topical administration ofcompositions containing vectors, see, e.g., U.S. Pat. No. 6,348,450regarding topical administration of vector compositions, and devices fortopical administration of compositions to wild or non-domesticatedanimals, see, e.g., WO01/95715, U.S. application Ser. No. 10/374,627,filed Feb. 26, 2003, for such devices for rodents and birds; each ofwhich, together with each document cited or referenced therein, as witheach document cited herein and each document referenced or cited in eachdocument cited herein, is hereby incorporated herein by reference.

The invention further relates to means and methods that makedifferential diagnosis possible, e.g., methods that make it possible tomake, or allow for, a distinction between an animal infected by the WestNile (WN) pathogenic virus and an animal administered a vaccine orimmunogenic composition according to the invention.

In certain embodiments, the invention provides in vitro and/or in vivoexpression vectors comprising a polynucleotide encoding the envelopeprotein E of WNV. In addition to the sources otherwise set forth hereinfor nucleic acid molecules encoding WNV E, mention is made of WO02/072036, published Sep. 19, 2002, with claims of priority to U.S.Provisional application Ser. Nos. 60/281,947 and 60/275,025, filed Apr.5, 2001 and Mar. 12, 2001, respectively. These vectors advantageouslyalso comprise the elements for the expression of the polynucleotide in ahost cell.

In addition to the polynucleotide encoding E, the expression vectorsaccording to the invention can comprise one or more otherpolynucleotides encoding other proteins of the WN virus, preferablystructural proteins of the WN virus and said sequences are preferablychosen from among those encoding the pre-membrane protein prM and themembrane protein M.

The vector preferably comprises a polynucleotide forming a singleencoding frame or coding region corresponding e.g. to prM-E, M-E, oradvantageously prM-M-E, or epitopes thereof; that is, expression of apolyprotein or epitopes thereof are considered advantageous. A vectorcomprising several separate polynucleotides encoding the differentproteins (e.g. prM and/or M and E or epitopes thereof) also falls withinthe scope of the present invention. The vector, especially for in vivoexpression, can also comprise polynucleotides corresponding to more thanone WN virus strain or isolate, for instance, two or morepolynucleotides encoding E or prM-M-E, or epitope(s) thereof, ofdifferent strains.

Likewise, an immunogenic or vaccine composition can comprise one or morevectors for expression of polynucleotides corresponding to more than oneWN virus strain or isolate, for instance, two or more polynucleotidesencoding E or prM-M-E, or epitope(s) thereof, of different strains. Thevector, especially for in vivo expression, can additionally comprise oneor more nucleotide sequences encoding immunogens of other pathogenicagents and/or cytokines.

According to a preferred embodiment of the invention, the expressionvector comprises a polynucleotide encoding prM-M-E and preferably in asingle reading frame. In this regard, and particularly in regard to theherein preference for E, prM, M and combinations thereof in view of thisdisclosure also acknowledging other WNV proteins, it is noted that inthis disclosure and particularly in the claims, terms such as“comprises”, “comprised”, “comprising” and the like can have the meaningattributed to it in U.S. patent law; e.g., they can mean “includes”,“included”, “including”, and the like; and that terms such as“consisting essentially of” and “consists essentially of” have themeaning ascribed to them in U.S. patent law, e.g., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of theinvention. It is further noted that in combinations or polyproteins, itis advantageous that E be among the structural proteins of thecombination or polyprotein.

The term polynucleotide encoding a protein of the WN virus primarilymeans a DNA fragment or isolated DNA molecule encoding said protein, orthe complementary strand thereto; but, RNA is not excluded, as it isunderstood in the art that thymidine (T) in a DNA sequence is consideredequal to uracil (U) in an RNA sequence. Thus, RNA sequences for use inthe invention, e.g., for use in RNA vectors, can be derived from DNAsequences, by thymidine (T) in the DNA sequence being considered equalto uracil (U) in RNA sequences.

The term protein includes peptides and polypeptides. A protein fragmentis immunologically active in the sense that once administered to thehost, it is able to evoke an immune response of the humoral and/orcellular type directed against the protein. Preferably the proteinfragment is such that it has substantially the same immunologicalactivity as the total protein. Thus, a protein fragment according to theinvention comprises or consists essentially of or consists of at leastone epitope or antigenic determinant. The term epitope relates to aprotein site able to induce an immune reaction of the humoral type (Bcells) and/or cellular type (T cells).

Accordingly, a minimum structure of the polynucleotide is that itcomprises or consists essentially of or consists of nucleotides toencode an epitope or antigenic determinant of the WNV protein orpolyprotein. A polynucleotide encoding a fragment of the total proteinor polyprotein, more advantageously, comprises or consists essentiallyof or consists of a minimum of 21 nucleotides, advantageously at least42 nucleotides, and preferably at least 57, 87 or 150 consecutive orcontiguous nucleotides of the sequence encoding the total protein orpolyprotein. As mentioned earlier, epitope determination procedures,such as, generating overlapping peptide libraries (Hemmer B. et al.,Immunology Today, 1998, 19 (4), 163-168), Pepscan (Geysen H. M. et al.,Proc. Nat. Acad. Sci. USA, 1984, 81 (13), 3998-4002; Geysen H. M. etal., Proc. Nat. Acad. Sci. USA, 1985, 82 (1), 178-182; Van der Zee R. etal., Eur. J. Immunol., 1989, 19 (1), 43-47; Geysen H. M., SoutheastAsian J. Trop. Med. Public Health, 1990, 21 (4), 523-533; Multipin®Peptide Synthesis Kits de Chiron) and algorithms (De Groot A. et al.,Nature Biotechnology, 1999, 17, 533-561), can be used in the practice ofthe invention, without undue experimentation. Other documents cited andincorporated herein may also be consulted for methods for determiningepitopes of an immunogen or antigen and thus nucleic acid molecules thatencode such epitopes.

In an advantageous embodiment, the polynucleotides according to theinvention comprise or consist essentially of or consist of thenucleotide sequence encoding one or two transmembrane domains andpreferably two of them, located in the terminal part C of the E proteinof WNV. For the WNV NY99 strain, these domains correspond to amino acidsequences 742 to 766 and 770 to 791 of GenBank AF196835.

Elements for the expression of the polynucleotide or polynucleotides areadvantageously present in an inventive vector. In minimum manner, thiscomprises, consists essentially of, or consists of an initiation codon(ATG), a stop codon and a promoter, and optionally also apolyadenylation sequence for certain vectors such as plasmid and certainviral vectors, e.g., viral vectors other than poxviruses. When thepolynucleotide encodes a polyprotein fragment, e.g. prM-E, M-E, prM-M-E,advantageously, in the vector, an ATG is placed at 5′ of the readingframe and a stop codon is placed at 3′. Other elements for controllingexpression may be present, such as enhancer sequences, stabilizingsequences and signal sequences permitting the secretion of the protein.

Methods for making and/or administering a vector or recombinants orplasmid for expression of gene products of genes of the invention eitherin vivo or in vitro can be any desired method, e.g., a method which isby or analogous to the methods disclosed in, or disclosed in documentscited in: U.S. Pat. Nos. 6,130,066, 5,494,807, 5,514,375, 5,744,140,5,744,141, 5,756,103, 5,762,938, 5,766,599, 5,990,091, 6,004,777,6,130,066, 6,497,883, 6,464,984, 6,451,770, 6,391,314, 6,387,376,6,376,473, 6,368,603, 6,348,196, 6,306,400, 6,228,846, 6,221,362,6,217,883, 6,207,166, 6,207,165, 6,159,477, 6,153,199, 6,090,393,6,074,649, 6,045,803, 6,033,670, 6,485,729, 6,103,526, 6,224,882,6,312,682, 6,312,683, 6,348,450, 4,603,112; 4,769,330; 5,174,993;5,505,941; 5,338,683; 5,494,807; 4,394,448; 4,722,848; 4,745,051;4,769,331; 5,591,639; 5,589,466; 4,945,050; 5,677,178; 5,591,439;5,552,143; and 5,580,859; U.S. patent application Ser. No. 920,197,filed Oct. 16, 1986; WO 94/16716; WO 96/39491; WO91/11525; WO 98/33510;WO 90/01543; EP 0 370 573; EP 265785; Paoletti (1996) Proc. Natl. Acad.Sci. USA 93:11349-11353; Moss (1996) Proc. Natl. Acad. Sci. USA93:11341-11348; Richardson (Ed) (1995) Methods in Molecular Biology 39,“Baculovirus Expression Protocols,” Humana Press Inc.; Smith et al.(1983) Mol. Cell. Biol. 3:2156-2165; Pennock et al. (1984) Mol. Cell.Biol. 4:399-406; Roizman Proc. Natl. Acad. Sci. USA 93:11307-11312;Andreansky et al. Proc. Natl. Acad. Sci. USA 93:11313-11318; Robertsonet al. Proc. Natl. Acad. Sci. USA 93:11334-11340; Frolov et al. Proc.Natl. Acad. Sci. USA 93:11371-11377; Kitson et al. (1991) J. Virol.65:3068-3075; Grunhaus et al. (1992) Sem. Virol. 3:237-52; Ballay et al.(1993) EMBO J. 4:3861-65; Graham (1990) Tibtech 8:85-87; Prevec et al.J. Gen. Virol. 70.429-434; Felgner et al. (1994) J. Biol. Chem.269:2550-2561; (1993) Science 259:174549; McClements et al. (1996) Proc.Natl. Acad. Sci. USA 93:11414-11420; Ju et al. (1998) Diabetologia41:736-739; and Robinson et al. (1997) Sem. Immunol. 9:271. Thus, thevector in the invention can be any suitable recombinant virus or virusvector, such as a poxvirus (e.g., vaccinia virus, avipox virus,canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.),adenovirus (e.g., canine adenovirus), herpesvirus, baculovirus,retrovirus, etc. (as in documents incorporated herein by reference); orthe vector can be a plasmid. The herein cited and incorporated herein byreference documents, in addition to providing examples of vectors usefulin the practice of the invention, can also provide sources for non-WNVproteins or epitopes thereof, e.g., non-WNV immunogens or epitopesthereof, cytokines, etc. to be expressed by vector or vectors in, orincluded in, multivalent or cocktail immunogenic compositions orvaccines of the invention.

The present invention also relates to preparations comprising vectors,such as expression vectors, e.g., vaccines or immunogenic compositions.The preparations can comprise, consist essentially of, or consist of oneor more vectors, e.g., expression vectors, such as in vivo expressionvectors, comprising, consisting essentially or consisting of (andadvantageously expressing) one or more of the WNV polynucleotidesencoding E, prM, M or combinations or polyproteins thereof, especiallyas above-mentioned (e.g., E, or E and prM, or E and M, or E and prM andM, or polyprotein E-prM-M, or polyprotein prM-E, or polyprotein M-E, orat least an epitope thereof); and, advantageously, the vector containsand expresses a polynucleotide that includes, consists essentially of,or consists of a coding region encoding WNV E, in a pharmaceutically orveterinarily acceptable carrier, excipient or vehicle. Thus, accordingto an embodiment of the invention, the other vector or vectors in thepreparation comprises, consists essentially of or consists of apolynucleotide that encodes, and under appropriate circumstances thevector expresses one or more other proteins of the WN virus, e.g. prM,M, prM-M, or an epitope thereof.

According to another embodiment, the vector or vectors in thepreparation comprise, or consist essentially of, or consist ofpolynucleotide(s) encoding one or more proteins or epitope(s) thereof ofWNV, e.g., of one or more WN virus strains or isolates; and,advantageously, in a suitable host cell or under appropriate conditions,the vector or vectors have express of the polynucleotide(s). Theinventive preparation advantageously comprises, consists essentially of,or consists of, at least two vectors comprising, consisting essentiallyof, or consisting of, and advantageously also expressing, preferably invivo under appropriate conditions or suitable conditions or in asuitable host cell, polynucleotides from different WN strains orisolates encoding the same proteins and/or for different proteins, butpreferably for the same proteins. As to preparations containing one ormore vectors containing, consisting essentially of or consisting ofpolynucleotides encoding, and preferably expressing, advantageously invivo, WNV E, or prM-M-E, or an epitope thereof, it is preferred that theexpression products be from two, three or more different WN strains orisolates, advantageously strains. The invention is also directed atmixtures of vectors that contain, consist essentially of, or consist ofcoding for, and express, prM, M, E, prM-M, prM-E or M-E of differentstrains. It is preferred that in such mixtures, at least one vectorcontain, consist essentially of, or consist of, coding for, and express,E.

According to yet another embodiment and as will be shown in greaterdetail hereinafter, the other vector or vectors in the preparationcomprise and express one or more cytokines and/or one or more immunogensof one or more other pathogenic agents. Sources for cytokines,immunogens for other pathogenic agents or epitope(s) thereof, andnucleic acid molecules encoding the same, may be found in herein citeddocuments, as well as in, WO02096349, WO0208162, WO0020025, WO00152888,WO0145735, WO00127097, WO0116330, WO0077210, WO0077188, WO0077043,WO9842743, WO9833928, WO9749826, WO9749825, U.S. Pat. Nos. 6,387,376,6,306,400, 6,159,477, 6,156,567, 6,153,199, 6,090,393, 6,074,649,6,033,670.

The invention also relates to various combinations of differentembodiments herein disclosed, e.g., compositions or vaccines containingvarious vectors, compositions or vaccines containing a vector and aprotein (WNV and/or non-WNV) and/or cytokine, etc.

The preparations comprising an in vitro or in vivo expression vectorcomprising and expressing a polynucleotide encoding prM-M-E constitute apreferred embodiment of the invention. According to another advantageousembodiment of the invention, the in vivo or in vitro expression vectorscomprise as the sole polynucleotide or polynucleotides of the WN virus,a polynucleotide encoding the protein E, optionally associated with prMand/or M, preferably encoding prM-M-E and optionally a signal sequenceof the WN virus. Thus, in advantageous embodiments the polynucleotidecan additionally encode a signal sequence of WNV.

According to a further advantageous embodiment, one or more of thenon-structural proteins NS2A, NS2B and NS3 are expressed jointly withthe structural proteins according to the invention, either via the sameexpression vector, or via their own expression vector. They arepreferably expressed together on the basis of a single polynucleotide,e.g., as a polyprotein. That is, in certain embodiments, the vectorfurther contains, consists essentially of or consists of, one or morenucleotides encoding NS2A, NS2B and/or NS3, or a composition or vaccinefurther contains, consists essentially of or consists of one or moreadditional vectors that contains, consists essentially of or consistsof, one or more nucleotides encoding NS2A, NS2B and/or NS3; this vectoror these vectors advantageously express(es) the non-structuralprotein(s); and, NS2A, NS2B and NS3 are advantageously expressedjointly, and more advantageously, as a polyprotein.

Thus, the invention also relates to vector such as an in vivo or invitro expression vector comprising, consisting essentially of orconsisting of the polynucleotide(s) encoding NS2A, NS2B, NS3,combinations thereof, including polyproteins thereof, such asNS2A-NS2B-NS3. The vector can be one of the above-described vectorscomprising, consisting essentially of or consisting of a polynucleotideencoding one or more structural proteins, e.g., E, prM, M, combinationsand polyproteins thereof such as prM-E, M-E, or prM-M-E, e.g., such avector that contains or consists essentially of polynucleotides encodingstructural protein or proteins or epitopes thereof can also contain orconsist essentially thereof polynucleotides encoding one or morenon-structural proteins, combination thereof, polyproteins thereof, orepitopes thereof. As an alternative, the invention relates to apreparation as described hereinbefore, also incorporating at least oneof the vectors that contain polynucleotide(s) encoding andadvantageously expressing a non-structural protein and optionally apharmaceutically or veterinarily acceptable carrier, vehicle orexcipient.

For preparing vectors, e.g., expression vectors, according to theinvention, the skilled artisan has available various strains of the WNvirus and the description of the nucleotide sequence of their genome,see, e.g., discussion herein and Savage H. M. et al. (Am. J. Trop. Med.Hyg. 1999, 61 (4), 600-611), table 2, which refers to 24 WN virusstrains and gives access references to polynucleotide sequences inGenBank, as well as other herein cited and incorporated by referencedocuments.

Reference is, for example, made to strain NY99 (GenBank AF196835). InGenBank, for each protein the corresponding DNA sequence is given(nucleotides 466-741 for prM, 742-966 for M, 967-2469 for E, or 466-2469for prM-M-E, 3526-4218 for NS2A, 4219-4611 for NS2B and 4612-6468 forNS3, or 3526-6468 for NS2A-NS2B-NS3). By comparison and alignment of thesequences, the determination of a polynucleotide encoding such a proteinin another WNV strain is readily determined.

As discussed herein, the term polynucleotide is understood to mean anucleic acid sequence encoding a protein or a fragment thereof or anepitope thereof specific to a particular WN virus; and, by equivalence,the term polynucleotide is understood to include the correspondingnucleotide sequences of the different WN virus strains and nucleotidesequences differing by due to codon degeneracy. Thus, a polynucleotideencoding WNV E is understood as comprising, consisting essentially of orconsisting of (a) nt 466-2469 of NY99 (GenBank AF196835), (b)corresponding sequences of different WNV strains, and (c) nucleotidesequences that encode WNV E but differ from (a) and (b) due to codondegeneracy.

Within the family of WN viruses, identity between amino acid sequences(“sequence identity”) prM-M-E relative to that of NY99 is equal to orgreater than 90%. Thus, the invention covers polynucleotides encodingproteins having amino acid sequences, whose sequence identity orhomology with the native WNV amino acid sequence for the protein isequal to or greater than 90%, advantageously 92%, preferably 95% andmore specifically 98%. For instance, an expressed E protein can havegreater than 90% identity with the sequence of the polypeptide expressedfrom (a) nt 466-2469 of NY99 (GenBank AF196835), (b) correspondingsequences of different WNV strains, and/or (c) nucleotide sequences thatencode WNV E but differ from (a) and (b) due to codon degeneracy;advantageously at least 92%, more advantageously at least 95%, and evenmore advantageously at least 98%.

Therefore, the invention comprehends polynucleotides that express suchhomologous polypeptides; and the corresponding degrees of homology oridentity of those polynucleotides to polynucleotides encodingpolypeptides to which homologous polypeptides have homology or identity.Homologous polypeptides advantageously contain one or more epitopes ofthe polypeptide to which there is identity or homology, such thathomologous polypeptides exhibit immunological similarity or identity tothe polypeptide to which there is identity or homology, e.g., thehomologous polypeptide elicits similar or better immune response (to theskilled immunologist) than polypeptide to which there is identity orhomology and/or the homologous polypeptide binds to antibodies elicitedby and/or to which the polypeptide to which there is identity orhomology binds, advantageously and not to other antibodies.

Accordingly, fragments of homologous polypeptides and of polypeptides towhich there is identity or homology, advantageously those fragmentswhich exhibit immunological similarity or identity to homologouspolypeptides or polypeptides to which there is identity or homology, areenvisioned as being expressed, and therefore, polynucleotides thereforwhich may represent fragments of polynucleotides of homologouspolypeptides and of polypeptides to which there is identity or homology,are also envisioned by and useful in the instant invention.

The term “sequence identity” indicates a quantitative measure of thedegree of homology between two amino acid sequences of equal length orbetween two nucleotide sequences of equal length. If the two sequencesto be compared are not of equal length, they must be aligned to bestpossible fit possible with the insertion of gaps or alternatively,truncation at the ends of the protein sequences. The sequence identitycan be calculated as ((N_(ref)−N_(dif))/N_(ref))×100, wherein N_(dif) isthe total number of non-identical residues in the two sequences whenaligned and wherein N_(ref) is the number of residues in one of thesequences. Hence, the DNA sequence AGTCAGTC will have a sequenceidentity of 75% with the sequence AATCAATC(N_(dif)=2 and N_(ref)=8). Agap is counted as non-identity of the specific residue(s), i.e. the DNAsequence AGTGTC will have a sequence identity of 75% with the DNAsequence AGTCAGTC (N_(dif)=2 and N_(ref)=8). Sequence identity canalternatively be calculated by the BLAST program e.g. the BLASTP program(Pearson W. R and D. J. Lipman (1988) PNAS USA 85:2444-2448)(www.ncbi.nlm.nih.gov/cgi-bin/BLAST). In one aspect of the invention,alignment is performed with the sequence alignment method ClustalW withdefault parameters as described by Thompson J., et al 1994, available athttp://www2.ebi.ac.uk/clustalw/. Thus, a polynucleotide can be anynucleic acid molecule including DNA, RNA, LNA (locked nucleic acids),PNA, RNA, dsRNA, RNA-DNA-hybrid, and non-naturally occurringnucleosides.

And from the herein disclosure, advantageously, proteins or polypeptidesexpressed by vectors of the invention are immunologically activepeptides and polypeptides, e.g., with respect to polypeptides orproteins of NY99, proteins or polypeptides expressed by vectors of theinvention can be:

a) corresponding proteins or polypeptides of one or more different WNvirus strains or isolates,

b) proteins differing therefrom (from NY99 and/or a)), but maintainingwith a native WN protein an identity equal to or greater than 90%,advantageously greater than or equal to 92%, more advantageously greaterthan or equal to 95% and even more advantageously greater than or equalto 98%.

Thus, a reference to a WNV protein may involve additional proteins asherein discussed.

Different WN virus strains are accessible in collections, especially inthe American Type Culture Collection (ATCC), e.g. under access numbersVR-82 or VR-1267, and as otherwise herein discussed, with it noted thatthe Kunjin virus is considered to be a WN virus.

In the invention, preferably the polynucleotide also comprises anucleotide sequence encoding a signal peptide, located upstream of thecoding for the expressed protein to facilitate the secretion thereof;and accordingly, the invention comprehends the expression of a WNVpolypeptide, such as a WNV antigen, immunogen, or fragment thereof,e.g., epitope, with a leader or signal sequence. The leader or signalsequence can be an endogenous sequence, e.g. the natural signal sequenceof a WNV polypeptide, which can be from the same WN virus strain orisolate or another strain or isolate. For example, for the NY99 WNvirus, the endogenous signal sequence for E is encoded at nucleotides922 to 966 of the GenBank sequence and for prM it is encoded atnucleotides 421 to 465. The leader or signal sequence can also be aheterologous sequence, and thus encoded by a nucleotide sequence that isheterologous to WNV. For example, the leader or signal sequence can beendogenous to the vector, or a leader or signal sequence that isheterologous to both the vector and WNV, such as a signal peptide oftissue plasminogen activator (tPA), e.g., human tPA, and thus, thevector or the polynucleotide therein can include a sequence encoding theleader or signal peptide, e.g., the leader or signal peptide of humantissue plasminogen activator (tPA) (Hartikka J. et al., Human GeneTherapy, 1996, 7, 1205-1217). The nucleotide sequence encoding thesignal peptide is advantageously inserted in frame and upstream of thesequence encoding the WNV polypeptide, e.g., E or its combinations, e.g.prM-M-E, M-E, prM-E.

According to an embodiment of the invention, the vectors, e.g., in vivoexpression vectors, are viral vectors.

Viral vectors, e.g., viral expression vectors are advantageously:poxviruses, e.g. vaccinia virus or an attenuated vaccinia virus, (forinstance, MVA, a modified Ankara strain obtained after more than 570passages of the Ankara vaccine strain on chicken embryo fibroblasts; seeStickl H. and Hochstein-Mintzel V., Munch. Med. Wschr., 1971, 113,1149-1153; Sutter G. et al., Proc. Natl. Acad. Sci. U.S.A., 1992, 89,10847-10851; available as ATCC VR-1508; or NYVAC, see U.S. Pat. No.5,494,807, for instance, Examples 1 to 6 and et seq of U.S. Pat. No.5,494,807 which discuss the construction of NYVAC, as well as variationsof NYVAC with additional ORFs deleted from the Copenhagen strainvaccinia virus genome, as well as the insertion of heterologous codingnucleic acid molecules into sites of this recombinant, and also, the useof matched promoters; see also WO96/40241), avipox virus or anattenuated avipox virus (e.g., canarypox, fowlpox, dovepox, pigeonpox,quailpox, ALVAC or TROVAC; see, e.g. U.S. Pat. Nos. 5,505,941,5,494,807), swinepox, raccoonpox, camelpox, or myxomatosis virus;adenoviruses, such as avian, canine, porcine, bovine, humanadenoviruses; or herpes viruses, such as equine herpes virus (EHVserotypes 1 and 4), canine herpes virus (CHV), feline herpes virus(FHV), bovine herpes viruses (BHV serotypes 1 and 4), porcine herpesvirus (PRV), Marek's disease virus (MDV serotypes 1 and 2), turkeyherpes virus (HVT or MDV serotype 3), or duck herpes virus. When aherpes virus is used, the vector HVT is preferred for the vaccination ofthe avian species and the vector EHV for the vaccination of horses.

More generally in certain embodiments, it may be advantageous to match avector to a host, such as an equine virus, e.g., EHV to use in equines,or a vector that is an avian pathogen, such as fowlpox HVT, MDV or duckherpes to use in avians such as poultry or chickens, or a vector that isa bovine pathogen such as BHV to use in bovines such as cows, or avector that is a porcine pathogen such a porcine herpes virus to use inporcines, or a vector that is a canine pathogen such as canineadenovirus or canine herpes virus to use in canines such as dogs, avector that is a feline pathogen such as FHV to use in felines, as thismay allow for an immune response against the vector and thus provide animmune response against a pathogen of the host or target species inaddition to an immune response against WNV.

However, it is also noted that it can be advantageous that the vectornot be a natural pathogen of the host; for instance, so that the vectorcan have expression of the exogenous, e.g., WNV coding sequences, butwith limited or no replication; for example, the use of an avipox vectorin a mammalian host, as in U.S. Pat. No. 5,174,993. It is also notedthat the invention comprehends vaccines, immunological and immunogeniccompositions, with those terms being used in the sense attributed tothem in the art; see, e.g., documents cited herein, such as U.S. Pat.No. 6,497,883.

According to another embodiment of the invention, the poxvirus vector,e.g., expression vector, is a canarypox virus or a fowlpox virus vector,advantageously an attenuated canarypox virus or fowlpox virus. In thisregard, is made to the canarypox available from the ATCC under accessnumber VR-111. Attenuated canarypox viruses are described in U.S. Pat.No. 5,756,103 (ALVAC) and WO01/05934. Numerous fowlpox virus vaccinationstrains are also available, e.g. the DIFTOSEC CT strain marketed byMERIAL and the NOBILIS VARIOLE vaccine marketed by Intervet; and,reference is also made to U.S. Pat. No. 5,766,599 which pertains to theattenuated fowlpox strain TROVAC.

For information on poxviruses and how to generate recombinants thereofand how to administer recombinants thereof, the skilled artisan canrefer documents cited herein and to WO90/12882, e.g., as to vacciniavirus mention is made of U.S. Pat. Nos. 4,769,330, 4,722,848, 4,603,112,5,110,587, 5,494,807, and 5,762,938 inter alia; as to fowlpox, mentionis made of U.S. Pat. Nos. 5,174,993, 5,505,941 and U.S. Pat. No.5,766,599 inter alia; as to canarypox mention is made of U.S. Pat. No.5,756,103 inter alia; as to swinepox mention is made of U.S. Pat. No.5,382,425 inter alia; and, as to raccoonpox, mention is made ofWO00/03030 inter alia.

When the expression vector is a vaccinia virus, insertion site or sitesfor the polynucleotide or polynucleotides to be expressed areadvantageously at the thymidine kinase (TK) gene or insertion site, thehemagglutinin (HA) gene or insertion site, the region encoding theinclusion body of the A type (ATI); see also documents cited herein,especially those pertaining to vaccinia virus. In the case of canarypox,advantageously the insertion site or sites are ORF(s) C3, C5 and/or C6;see also documents cited herein, especially those pertaining tocanarypox virus. In the case of fowlpox, advantageously the insertionsite or sites are ORFs F7 and/or F8; see also documents cited herein,especially those pertaining to fowlpox virus. The insertion site orsites for MVA virus area advantageously as in various publications,including Carroll M. W. et al., Vaccine, 1997, 15 (4), 387-394;Stittelaar K. J. et al., J. Virol., 2000, 74 (9), 4236-4243; Sutter G.et al., 1994, Vaccine, 12 (11), 1032-1040; and, in this regard it isalso noted that the complete MVA genome is described in Antoine G.,Virology, 1998, 244, 365-396, which enables the skilled artisan to useother insertion sites or other promoters.

Preferably, when the expression vector is a poxvirus, the polynucleotideto be expressed is inserted under the control of a specific poxviruspromoter, e.g., the vaccinia promoter 7.5 kDa (Cochran et al., J.Virology, 1985, 54, 30-35), the vaccinia promoter I3L (Riviere et al.,J. Virology, 1992, 66, 3424-3434), the vaccinia promoter HA (Shida,Virology, 1986, 150, 451-457), the cowpox promoter ATI (Funahashi etal., J. Gen. Virol., 1988, 69, 35-47), the vaccinia promoter H6 (TaylorJ. et al., Vaccine, 1988, 6, 504-508; Guo P. et al. J. Virol., 1989, 63,4189-4198; Perkus M. et al., J. Virol., 1989, 63, 3829-3836), interalia.

Preferably, for the vaccination of mammals the expression vector is acanarypox or a fowlpox. In this way, there can be expression of theheterologous proteins, e.g., WNV proteins, with limited or no productivereplication. Preferably, for the vaccination of avians, e.g., chickens,ducks, turkeys and geese, the expression vector is a canarypox or afowlpox.

When the expression vector is a herpes virus of turkeys or HVT,advantageous insertion site or sites are located in the BamHI I fragmentor in the BamHI M fragment of HVT. The HVT BamHI I restriction fragmentcomprises several open reading frames (ORFs) and three intergene regionsand comprises several preferred insertion zones, such as the threeintergene regions 1, 2 and 3, which are preferred regions, and ORF UL55(see, e.g., FR-A-2 728 795, U.S. Pat. No. 5,980,906). The HVT BamHI Mrestriction fragment comprises ORF UL43, which is also a preferredinsertion site (see, e.g., FR-A-2 728 794, U.S. Pat. No. 5,733,554).

When the expression vector is an EHV-1 or EHV-4 herpes virus,advantageous insertion site or sites include TK, UL43 and UL45 (see,e.g., EP-A-668355). Preferably, when the expression vector is a herpesvirus, the polynucleotide to be expressed is inserted under the controlof a eukaryotic promoter, such as a strong eukaryote promoter,preferably a CMV-IE (murine or human) promoter; that is, in embodimentsherein, the polynucleotide to be expressed is operably linked to apromoter, and in herpes virus embodiments, advantageously thepolynucleotide to be expressed is operably linked to a strong eukaryoticpromoter such as a mCMV-IE or hCMV-IE promoter. Strong promoters arealso discussed herein in relation to plasmids as vectors.

According to a yet further embodiment of the invention, the vector,e.g., in vivo expression vector, is a plasmidic vectors, also known as aplasmid vector or a DNA plasmid vector, e.g., the type of plasmid vectoremployed in that which is known as a DNA vaccine (in contrast with atransfection plasmid used in homologous recombination to generate arecombinant virus, which is not used in a DNA vaccine).

The term plasmid covers any DNA transcription unit in the form of apolynucleotide sequence comprising a polynucleotide according to theinvention and the elements necessary for its in vivo expression of thatwhich is encoded by the polynucleotide in a cell or cells of the desiredhost or target; and, in this regard, it is noted that there is asupercoiled or non-supercoiled, circular plasmid, as well as linearforms, all of which are intended to be within the scope of theinvention.

Each plasmid comprises or contains or consists essentially of, inaddition to the polynucleotide encoding the antigen or epitope of thepathogen or pathogens, e.g., WNV (or WNV and another pathogen), apromoter for expression, in the host cells cor cells, of thepolynucleotide; and, the polynucleotide may be said to be operablylinked to the promoter or under the control of the promoter or dependentupon the promoter. In general, it is advantageous to employ a eukaryoticpromoter, e.g., a strong eukaryotic promoter. The preferred strongeukaryote promoter is the early cytomegalovirus promoter (CMV-IE) ofhuman or murine origin, or optionally having another origin such as therat or guinea pig. The CMV-IE promoter can comprise the actual promoterpart, which may or may not be associated with the enhancer part.Reference can be made to EP-A-260 148, EP-A-323 597, U.S. Pat. Nos.5,168,062, 5,385,839, and 4,968,615, as well as to PCT WO87/03905. TheCMV-IE promoter is preferably a human CMV-IE (Boshart M. et al., Cell.,1985, 41, 521-530) or murine CMV-IE.

In more general terms, the promoter has either a viral or a cellularorigin. A strong viral promoter other than CMV-IE that may be usefullyemployed in the practice of the invention is the early/late promoter ofthe SV40 virus or the LTR promoter of the Rous sarcoma virus. A strongcellular promoter that may be usefully employed in the practice of theinvention is the promoter of a gene of the cytoskeleton, such as e.g.the desmin promoter (Kwissa M. et al., Vaccine, 2000, 18 (22),2337-2344), or the actin promoter (Miyazaki J. et al., Gene, 1989, 79(2), 269-277).

Functional subfragments of these promoters, i.e., portions of thesepromoters that maintain an adequate promoting activity, are includedwithin the present invention, e.g. truncated CMV-IE promoters accordingto WO98/00166 or U.S. Pat. No. 6,156,567 can be used in the practice ofthe invention. A promoter in the practice of the invention consequentlyincludes derivatives and subfragments of a full-length promoter thatmaintain an adequate promoting activity and hence function as apromoter, preferably promoting activity substantially similar to that ofthe actual or full-length promoter from which the derivative orsubfragment is derived, e.g., akin to the activity of the truncatedCMV-IE promoters of U.S. Pat. No. 6,156,567 to the activity offull-length CMV-IE promoters. Thus, a CMV-IE promoter in the practice ofthe invention can comprise or consist essentially of or consist of thepromoter portion of the full-length promoter and/or the enhancer portionof the full-length promoter, as well as derivatives and subfragments.

Preferably, the plasmids comprise or consist essentially of otherexpression control elements. It is particularly advantageous toincorporate stabilizing sequence(s), e.g., intron sequence(s),preferably intron II of the rabbit β-globin gene (van Ooyen et al.,Science, 1979, 206: 337-344).

As to the polyadenylation signal (polyA) for the plasmids and viralvectors other than poxviruses, use can more be made of the polyA signalof the bovine growth hormone (bGH) gene (see U.S. Pat. No. 5,122,458),or the poly(A) signal of the rabbit β-globin gene or the poly(A) signalof the SV40 virus.

As to other expression control elements usable in plasmids, attention isdirected to expression control elements that are useful in herpes virusexpression vectors.

According to another embodiment of the invention, the expression vectorsare expression vectors used for the in vitro expression of proteins inan appropriate cell system. The expressed proteins can be harvested inor from the culture supernatant after, or not after secretion (if thereis no secretion a cell lysis typically occurs or is performed),optionally concentrated by concentration methods such as ultrafiltrationand/or purified by purification means, such as affinity, ion exchange orgel filtration-type chromatography methods.

Protein production can take place by the transfection of mammalian cellsby plasmids, by replication or expression without productive replicationof viral vectors on mammal cells or avian cells, or by Baculovirusreplication (see, e.g., U.S. Pat. No. 4,745,051; Vialard J. et al., J.Virol., 1990 64 (1), 37-50; Verne A., Virology, 1988, 167, 56-71), e.g.Autographa californica Nuclear Polyhedrosis Virus AcNPV, on insect cells(e.g. Sf9 Spodoptera frugiperda cells, ATCC CRL 1711; see also U.S. Pat.Nos. 6,228,846, 6,103,526). Mammalian cells which can be used areadvantageously hamster cells (e.g. CHO or BHK-21) or monkey cells (e.g.COS or VERO). Thus, the invention accordingly comprehends expressionvectors incorporating a polynucleotide according to the invention, aswell as the thus produced or expressed WNV proteins or fragments thereoffrom in vitro expression, and the preparations containing the same.

Accordingly, the present invention also relates to WNVprotein-concentrated and/or purified preparations. When thepolynucleotide encodes several proteins, they are cleaved, and theaforementioned preparations then contain cleaved proteins.

The present invention also relates to immunogenic compositions andvaccines against the WN virus comprising at least one in vivo expressionvector according to the invention and a pharmaceutically or veterinarilyacceptable excipient or carrier or vehicle, and optionally an adjuvant.

An immunogenic composition covers any composition which, onceadministered to the target species, induces an immune response againstthe WN virus. The term vaccine is understood to mean a composition ableto induce an effective protection. The target species include mammals,e.g., equines, canines, felines, bovines, porcines and humans; reptiles,and birds or avians; preferably horse, dog, cat, pig, alligator; and, inthe case of birds or avians, geese, turkeys, chickens and ducks. Thislist is meant to include reproducing animals, egg-laying animals,meat-producing animals or production animals (animals whose flesh iscommonly consumed by some humans), and companion animals (animals whoare kept as pets by humans).

The pharmaceutically or veterinarily acceptable carriers or vehicles orexcipients are well known to the one skilled in the art. For example, apharmaceutically or veterinarily acceptable carrier or vehicle orexcipient can be a 0.9% NaCl saline solution or a phosphate buffer. Thepharmaceutically or veterinarily acceptable carrier or vehicle orexcipients may be any compound or combination of compounds facilitatingthe administration of the vector (or protein expressed from an inventivevector in vitro); advantageously, the carrier, vehicle or excipient mayfacilitate transfection and/or improve preservation of the vector (orprotein). Doses and dose volumes are herein discussed in the generaldescription of immunization and vaccination methods, and can also bedetermined by the skilled artisan from this disclosure read inconjunction with the knowledge in the art, without any undueexperimentation.

The immunogenic compositions and vaccines according to the inventionpreferably comprise or consist essentially of one or more adjuvants.Particularly suitable adjuvants for use in the practice of the presentinvention are (1) polymers of acrylic or methacrylic acid, maleicanhydride and alkenyl derivative polymers, (2) immunostimulatingsequences (ISS), such as oligodeoxyribonucleotide sequences having oneor more non-methylated CpG units (Klinman D. M. et al., Proc. Natl.Acad. Sci., USA, 1996, 93, 2879-2883; WO98/16247), (3) an oil in wateremulsion, such as the SPT emulsion described on p 147 of “VaccineDesign, The Subunit and Adjuvant Approach” published by M. Powell, M.Newman, Plenum Press 1995, and the emulsion MF59 described on p 183 ofthe same work, (4) cation lipids containing a quaternary ammonium salt,(5) cytokines, (6) aluminum hydroxide or aluminum phosphate or (7) otheradjuvants discussed in any document cited and incorporated by referenceinto the instant application, or (8) any combinations or mixturesthereof.

The oil in water emulsion (3), which is especially appropriate for viralvectors, can be based on:

-   -   light liquid paraffin oil (European pharmacopoeia type),    -   isoprenoid oil such as squalane, squalene,    -   oil resulting from the oligomerization of alkenes, e.g.        isobutene or decene,    -   esters of acids or alcohols having a straight-chain alkyl group,        such as vegetable oils, ethyl oleate, propylene glycol,        di(caprylate/caprate), glycerol tri(caprylate/caprate) and        propylene glycol dioleate, or    -   esters of branched, fatty alcohols or acids, especially        isostearic acid esters.

The oil is used in combination with emulsifiers to form an emulsion. Theemulsifiers may be nonionic surfactants, such as:

-   -   esters of on the one hand sorbitan, mannide (e.g.        anhydromannitol oleate), glycerol, polyglycerol or propylene        glycol and on the other hand oleic, isostearic, ricinoleic or        hydroxystearic acids, said esters being optionally ethoxylated,    -   polyoxypropylene-polyoxyethylene copolymer blocks, such as        Pluronic, e.g., L121.

Among the type (1) adjuvant polymers, preference is given to polymers ofcrosslinked acrylic or methacrylic acid, especially crosslinked bypolyalkenyl ethers of sugars or polyalcohols. These compounds are knownunder the name carbomer (Pharmeuropa, vol. 8, no. 2, June 1996). Oneskilled in the art can also refer to U.S. Pat. No. 2,909,462, whichprovides such acrylic polymers crosslinked by a polyhydroxyl compoundhaving at least three hydroxyl groups, preferably no more than eightsuch groups, the hydrogen atoms of at least three hydroxyl groups beingreplaced by unsaturated, aliphatic radicals having at least two carbonatoms. The preferred radicals are those containing 2 to 4 carbon atoms,e.g. vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals can also contain other substituents, such asmethyl. Products sold under the name Carbopol (BF Goodrich, Ohio, USA)are especially suitable. They are crosslinked by allyl saccharose or byallyl pentaerythritol. Among them, reference is made to Carbopol 974P,934P and 971P.

As to the maleic anhydride-alkenyl derivative copolymers, preference isgiven to EMA (Monsanto), which are straight-chain or crosslinkedethylene-maleic anhydride copolymers and they are, for example,crosslinked by divinyl ether. Reference is also made to J. Fields etal., Nature 186: 778-780, Jun. 4, 1960.

With regard to structure, the acrylic or methacrylic acid polymers andEMA are preferably formed by basic units having the following formula:

in which:

-   -   R₁ and R₂, which can be the same or different, represent H or        CH₃    -   x=0 or 1, preferably x=1    -   y=1 or 2, with x+y=2.

For EMA, x=0 and y=2 and for carbomers x=y=1.

These polymers are soluble in water or physiological salt solution (20g/l NaCl) and the pH can be adjusted to 7.3 to 7.4, e.g., by soda(NaOH), to provide the adjuvant solution in which the expressionvector(s) can be incorporated. The polymer concentration in the finalvaccine composition can range between 0.01 and 1.5% w/v, advantageously0.05 to 1% w/v and preferably 0.1 to 0.4% w/v.

The cationic lipids (4) containing a quaternary ammonium salt which areadvantageously but not exclusively suitable for plasmids, are preferablythose having the following formula:

in which R₁ is a saturated or unsaturated straight-chain aliphaticradical having 12 to 18 carbon atoms, R₂ is another aliphatic radicalcontaining 2 or 3 carbon atoms and X is an amine or hydroxyl group.

Among these cationic lipids, preference is given to DMRIE(N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneammonium; WO96/34109), preferably associated with a neutral lipid,preferably DOPE (dioleoyl-phosphatidyl-ethanol amine; Behr J. P., 1994,Bioconjugate Chemistry, 5, 382-389), to form DMRIE-DOPE.

Preferably, the plasmid mixture with the adjuvant is formedextemporaneously and preferably contemporaneously with administration ofthe preparation or shortly before administration of the preparation; forinstance, shortly before or prior to administration, theplasmid-adjuvant mixture is formed, advantageously so as to give enoughtime prior to administration for the mixture to form a complex, e.g.between about 10 and about 60 minutes prior to administration, such asapproximately 30 minutes prior to administration.

When DOPE is present, the DMRIE:DOPE molar ratio is preferably about95:about 5 to about 5:about 95, more preferably about 1:about 1, e.g.,1:1.

The DMRIE or DMRIE-DOPE adjuvant:plasmid weight ratio can be betweenabout 50:about 1 and about 1:about 10, such as about 10:about 1 andabout 1:about 5, and preferably about 1:about 1 and about 1:about 2,e.g., 1:1 and 1:2.

The cytokine or cytokines (5) can be in protein form in the immunogenicor vaccine composition, or can be co-expressed in the host with theimmunogen or immunogens or epitope(s) thereof. Preference is given tothe co-expression of the cytokine or cytokines, either by the samevector as that expressing the immunogen or immunogens or epitope(s)thereof, or by a separate vector therefor.

The cytokine(s) can be chosen from: interleukin 18 (IL-18), interleukin12 (IL-12), interleukin 15 (IL-15), MIP-1α (macrophage inflammatoryprotein 1α; Marshall E. et al., Br. J. Cancer, 1997, 75 (12),1715-1720), GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor).Particular reference is made to avian cytokines, for instance, those ofthe chicken, such as cIL-18 (Schneider K. et al., J. Interferon CytokineRes., 2000, 20 (10), 879-883), cIL-15 (Xin K.-Q. et al., Vaccine, 1999,17, 858-866), and equine cytokines, for instance equine GM-CSF(WO00/77210). Preferably, use is made of cytokines of the species to bevaccinated; that is, advantageously, the cytokine is matched to thetarget or host species, and, note for example, canine GM-CSF (example 8of WO00/77043), feline GM-CSF (example 9 of WO00/77043).

WO00/77210 provides the nucleotide sequence and the amino acid sequencecorresponding to equine GM-CSF, the in vitro GM-CSF production and theconstruction of vectors (e.g., plasmids and viral vectors) permitting invivo equine GM-CSF expression. These proteins, plasmids and viralvectors can be used in immunogenic compositions and equine vaccinesaccording to the invention. For example, use can be made of the plasmidpJP097 described in example 3 of WO00/77210 or use can be made of theteaching of the latter in order to produce other vectors or for the invitro production of equine GM-CSF and the incorporation of the vectorsor the equine GM-CSF into immunogenic compositions or equine vaccinesaccording to the invention.

The present invention also relates to immunogenic compositions andso-called subunit vaccines, incorporating or comprising or consistingessentially of the protein E and optionally one or more other hereinmentioned proteins of the WN virus, e.g., prM or M and advantageouslyproduced by in vitro expression in the manner described herein, as wellas a pharmaceutically or veterinarily acceptable carrier or vehicle orexcipient.

The pharmaceutically or veterinarily acceptable carrier or vehicle orexcipient can be determined by the skilled artisan without undueexperimentation from the disclosure herein and the knowledge in the art,e.g., by reference to documents cited and incorporated herein ordocuments referenced in herein cited documents and incorporated hereinby reference; and, can for example, be 0.9% NaCl saline solution orphosphate buffer.

The immunogenic compositions and subunit vaccines according to theinvention preferably comprise or consist essentially of one or moreadjuvants. Especially suitable for use in the present invention are (1)an acrylic or methacrylic acid polymer, or a maleic anhydride andalkenyl derivative polymer, (2) an immunostimulating sequence (ISS),such as an oligodeoxyribonucleotide sequence having one or morenon-methylated CpG units (Klinman D. M. et al., Proc. Natl. Acad. Sci.USA, 1996, 93, 2879-2883; WO98/16247), (3) an oil in water emulsion,such as the emulsion SPT described on p 147 of “Vaccine Design, TheSubunit and Adjuvant Approach”, published by M. Powell, M. Newmann,Plenum Press 1995, and the emulsion MF59 described on p 183 of the samework, (4) a water in oil emulsion (EP-A-639 071), (5) saponin, such asQuil-A, or (6) alumina hydroxide or an equivalent. The different typesof adjuvants defined under 1), 2) and 3) have been described in greaterdetail herein in connection with the expression vector-based vaccinesand immunogenic compositions.

The doses and dose volumes are discussed herein in connection with thegeneral description of immunization and vaccination methods.

Animals immunized with immunogenic compositions or vaccines according tothe invention develop a specific immunity against WNV, which during aWNV infection involves a decrease of the viremia, and indeed can totallyblock the virus, as compared with unvaccinated control animals. Thisadvantageous aspect of the invention may be used to stop thetransmission of the WN virus, to limit the existence of viral reservoirsand to prevent outbreaks of West Nile disease, notably in human.

Another advantageous aspect of the invention is that protective immunitycan be transmitted from vaccinated subjects to the offspring.

According to the invention, the vaccination against the WN virus can becombined with other vaccinations within the framework of vaccinationprograms, in the form of immunization or vaccination kits or methods, orin the form of multivalent immunogenic compositions and multivalentvaccines, i.e. comprising or consisting essentially of at least onevaccine component against the WN virus and at least one vaccinecomponent against at least one other pathogenic agent. This alsoincludes the expression by the same expression vector of genes of atleast two pathogenic agents, including the WN virus.

The invention thus also relates to a multivalent or “cocktail”immunogenic composition or a multivalent or “cocktail” vaccine againstthe WN virus and against at least one other pathogen of the targetspecies, using the same in vivo expression vector containing andexpressing at least one polynucleotide of the WN virus according to theinvention and at least one polynucleotide expressing an immunogen ofanother pathogen. As to combination or multivalent or “cocktail”immunogenic compositions or vaccines, as well as to immunogens orantigens or epitopes thereof to be in or expressed by such compositionsor vaccines, attention is directed to herein cited and incorporated byreference documents, as well as to U.S. Pat. Nos. 5,843,456 and6,368,603.

The “immunogen” expressed by a vector of the invention or used inmultivalent or “cocktail” compositions or vaccines is understood to meana protein, glycoprotein, polypeptide, peptide, epitope or derivative,e.g. fusion protein, inducing an immune response, preferably of aprotective nature.

As discussed herein, these multivalent compositions or vaccines can alsocomprise or consist essentially of a pharmaceutically or veterinarilyacceptable carrier or vehicle or excipient, and optionally an adjuvant.

The invention also relates to a multivalent immunogenic composition or amultivalent vaccine comprising at least one in vivo expression vector inwhich at least one polynucleotide of the WN virus is inserted (andexpressed in vivo) and at least a second expression vector in which apolynucleotide encoding an immunogen of another pathogenic agent isinserted (and expressed in vivo). Such multivalent compositions orvaccines also comprise or consist essentially of a pharmaceutically orveterinarily acceptable carrier or vehicle or excipient, and optionallyan adjuvant.

For antigen(s) or immunogen(s) or epitope(s) to be included in orexpressed by a multivalent immunogenic composition or vaccine (inaddition to WNV antigen(s), immunogen(s) or epitope(s)), including as todetermining or ascertaining epitope(s), the skilled artisan may consultherein cited documents and documents cited in herein cited documents,all of which are incorporated by reference into the instant application.

For equine multivalent immunogenic compositions and multivalentvaccines, the additional equine pathogen(s), as to which additionalequine antigen(s) or immunogen(s) or epitope(s) thereof are included inand/or expressed by the multivalent immunogenic compositions andmultivalent vaccines, are advantageously chosen from among the groupincluding viruses of equine rhinopneumonia, EHV-1 and/or EHV-4 (andpreferably there is a combination of immunogens of EHV-1 and EHV-4),equine influenza virus, EIV, eastern encephalitis virus, EEV, westernencephalitis virus, WEV, Venezuelan encephalitis virus, VEV (preferencebeing given to a combination of the three, i.e., EEV, WEV and VEV),Clostridium tetani (tetanus), and mixtures thereof. Preferably, for EHVthe immunogen is gB and/or gD see also U.S. Pat. Nos. 6,395,283,6,248,333, 5,338,683, 6,183,750; for herpesvirus immunogens andconstructs expressing the same); for EIV the immunogen is advantageouslyHA, NP and/or N; for viruses of encephalitis, the immunogen isadvantageously C and/or E2; and for Clostridium tetani the immunogen isall or part of the subunit C of the tetanic toxin. Thus, the inventioncomprehends the use of polynucleotide(s) encoding (an) immunologicallyactive fragment(s) or (an) epitope(s) of such immunogen(s).

For canine multivalent immunogenic compositions and multivalentvaccines, the additional canine pathogen(s), as to which additionalcanine antigen(s) or immunogen(s) or epitope(s) thereof are included inand/or expressed by the multivalent immunogenic compositions andmultivalent vaccines, are advantageously chosen from among the groupincluding viruses of measles disease virus, canine distemper virus(CDV), canine parainfluenza type 2 virus (CPI-2), canine herpesvirustype 1 (CHV-1), rabies virus (rhabdovirus), canine parvovirus (CPV),canine coronavirus (CCV), canine adenovirus, Borrelia burgdorferi,Leptospira and mixtures thereof. Preferably, for CDV the immunogen isadvantageously F and/or HA (see also U.S. Pat. Nos. 6,309,647, 5,756,102regarding CDV immunogens and constructs); for CPV the immunogen isadvantageously VP2; for CCV the immunogen is advantageously S and/or M;for CHV-1 the immunogen is advantageously gB and/or gC and/or gD (seealso U.S. Pat. Nos. 5,688,920, 5,529,780, regarding CHV immunogens andconstructs); for rabies virus the immunogen is advantageously G (seealso U.S. Pat. No. 5,843,456 regarding rabies combination compositions);for Borrelia burgdorferi the immunogen is advantageously OspA (see alsoU.S. Pat. No. 6,368,603 regarding OspA combination compositions). Theinvention thus comprehends the use of polynucleotide(s) encoding (an)immunologically active fragment(s) or an epitope(s) of suchimmunogen(s).

For feline multivalent immunogenic compositions and multivalentvaccines, the additional feline pathogen(s), as to which additionalfeline antigen(s) or immunogen(s) or epitope(s) thereof are included inand/or expressed by the multivalent immunogenic compositions andmultivalent vaccines, are advantageously chosen from among the groupincluding viruses of the feline herpesvirus type 1 (FHV-1), felinecalicivirus (FCV), rabies virus (rhabdovirus), feline parvovirus (FPV),feline infectious peritonitis virus (FIPV), feline leukaemia virus(FeLV), feline immunodeficiency virus (FIV), Chlamydia and mixturesthereof. Preferably, for FeLV the immunogen is advantageously A and/or Band/or gag and/or pol, e.g., gag/pol; for FPV the immunogen isadvantageously VP2; for FIPV the immunogen is advantageously S and/or Mand/or N, e.g., S and M and/or N (see also U.S. Pat. Nos. 6,348,196 and5,858,373 and immunogens and constructs thereof); for FHV the immunogenis advantageously gB and/or gC and/or gD, e.g., gB and gC and/or gD (seealso U.S. Pat. Nos. 5,338,683, 6,183,750; for herpesvirus immunogens andconstructs expressing the same); for FCV the immunogen is advantageouslyC; for FIV the immunogen is advantageously env and/or gag and/or pro,e.g., gag/pro, env, or env and gag/pro (see also immunogens andconstructs discussed in Tartaglia et al., U.S. application Ser. No.08/746,668, filed Nov. 14, 1996); for rabies virus the immunogen isadvantageously G. The invention thus comprehends the use ofpolynucleotide(s) encoding (an) immunologically active fragment(s) or(an) epitope(s) of said immunogen(s).

For avian multivalent immunogenic compositions and multivalent vaccines,the additional avian pathogen(s), as to which additional avianantigen(s) or immunogen(s) or epitope(s) thereof are included in and/orexpressed by the multivalent immunogenic compositions and multivalentvaccines, are advantageously chosen from among the group includingviruses of the Marek's disease virus (MDV) (e.g., serotypes 1 and 2,preferably 1), Newcastle disease virus (NDV), Gumboro disease virus orinfectious bursal disease virus (IBDV), infectious bronchitis virus(IBV), infectious anaemia virus or chicken anemia virus (CAV),infectious laryngotracheitis virus (ILTV), encephalomyelitis virus oravian encephalomyelitis virus (AEV or avian leukosis virus ALV), virusof hemorragic enteritis of turkeys (HEV), pneumovirosis virus (TRTV),fowl plague virus (avian influenza), chicken hydropericarditis virus,avian reoviruses, Escherichia coli, Mycoplasma gallinarum, Mycoplasmagallisepticum, Haemophilus avium, Pasteurella gallinarum, Pasteurellamultocida gallicida, and mixtures thereof. Preferably, for MDV theimmunogen is advantageously gB and/or gD, e.g., gB and gD, for NDV theimmunogen is advantageously FIN and/or F, e.g., FIN and F; for IBDV theimmunogen advantageously is VP2; for IBV the immunogen is advantageouslyS (more advantageously S1) and/or M and/or N, e.g., S (or S1) and Mand/or N; for CAV the immunogen is advantageously VP1 and/or VP2; forILTV the immunogen is advantageously gB and/or gD; for AEV the immunogenadvantageously is env and/or gag/pro, e.g., env and gag/pro or gag/pro;for HEV the immunogen is advantageously the 100K protein and/or hexon;for TRTV the immunogen is advantageously F and/or G, and for fowl plaguethe immunogen is advantageously HA and/or N and/or NP, e.g., HA and Nand/or NP. The invention thus comprehends the use of polynucleotide(s)encoding (an) immunologically active fragment(s) or (an) epitope(s) ofsaid immunogen(s).

By way of example, in a multivalent immunogenic composition or amultivalent vaccine according to the invention, to which one or moreadjuvants has optionally been added (and hence the composition containsor consists essentially of or consists of one or more adjuvants) asdiscussed herein, and which is intended for equine species, it ispossible to incorporate (and hence for the composition or vaccine tocomprise, consist essentially of or consist of) one or more of theplasmids described in WO98/03198, advantageously as discussed inexamples 8 to 25 thereof, and/or those described in WO00/77043 and whichrelate to the equine species, advantageously those described in examples6 and 7 thereof. For the canine species, a multivalent composition orvaccine may contain or consist essentially of or consist of one or moreof the plasmids described in WO98/03199, advantageously as discussed inexamples 8 to 16 thereof, and/or those described in WO00/77043 and whichrelate to the canine species, advantageously those described in examples2, 3 and 4 thereof; and, such compositions or vaccines can contain,consist essentially of or consist of one or more adjuvants. For thefeline species, a multivalent composition or vaccine may contain orconsist essentially of or consist of one or more of the plasmidsdescribed in WO98/03660, advantageously in examples 8 to 19 thereof,and/or those described in WO00/77043 and which relate to the felinespecies, advantageously those described in example 5 thereof; and, suchcompositions or vaccines can contain, consist essentially of or consistof one or more adjuvants. And for the avian species, a multivalentcomposition or vaccine may contain or consist essentially of or consistof one or more of the plasmids described in WO98/03659, advantageouslyin examples 7 to 27 thereof; and, such compositions or vaccines cancontain, consist essentially of or consist of one or more adjuvants.

The immunogenic compositions or vaccines as discussed herein can also becombined with at least one conventional vaccine (e.g., inactivated, liveattenuated, or subunit) directed against the same pathogen or at leastone other pathogen of the species to which the composition or vaccine isdirected. The immunogenic compositions or vaccines discussed herein canbe administered prior to or after the conventional vaccine, e.g., in a“prime-boost” regimen.

The invention further comprehends combined vaccination employingimmunogenic composition(s) and subunit vaccine(s) according to theinvention. Thus, the invention also relates to multivalent immunogeniccompositions and multivalent vaccines comprising one or more proteinsaccording to the invention and one or more immunogens (as the termimmunogen is discussed herein) of at least one other pathogenic agent(advantageously from among those herein and in documents cited andincorporated herein by reference) and/or another pathogenic agent ininactivated or attenuated form or as a subunit. In the manner described,these multivalent vaccines or compositions also contain, consistessentially of or consist of a pharmaceutically or veterinarilyacceptable vehicle or excipient and optionally one or more adjuvants.

The present invention also relates to methods for the immunization andvaccination of a target species, e.g., as discussed herein.

The present invention also relates to methods for the immunizationand/or vaccination of a target species, using a prime-boost regimen. Theterm of “prime-boost” refers to the successive administrations of twodifferent vaccine types or immunogenic or immunological compositiontypes having at least one immunogen in common. The primingadministration (priming) is the administration of a first vaccine orimmunogenic or immunological composition type and may comprise one, twoor more administrations. The boost administration is the administrationof a second vaccine or immunogenic or immunological composition type andmay comprise one, two or more administrations, and, for instance, maycomprise or consist essentially of annual administrations.

An embodiment of a prime-boost immunization or vaccination against WNVaccording to the invention is a prime-boost immunization or vaccinationwherein the animal is first administered a (priming) DNA vaccine orimmunological or immunogenic composition comprising or consistingessentially of and expressing in vivo at least one immunogen, antigen orepitope of WNV, and thereafter is administered (boosted with) a secondtype of vaccine or immunogenic or immunological composition containingor consisting essentially of or expressing at least one immunogen,antigen or epitope that is common to the priming vaccine or immunogenicor immunological composition. This second type of vaccine can be a aninactivated, or attenuated or subunit vaccine or immunogenic orimmunological composition or a vector, e.g., recombinant or modifiedvirus vaccine or immunogenic or immunological composition that has invivo expression (e.g. poxvirus, herpesvirus, adenovirus). Poxviruses maybe advantageously employed, e.g., attenuated vaccinia viruses, like MVAor NYVAC, and avipox viruses, like canarypox viruses and fowlpoxviruses.

Advantageously, the DNA vaccine is intended to induce a priming immuneresponse specific for the expressed immunogen, antigen or epitope or“DNA induced immune response” (such as a gamma-interferon+ (IFN_(γ)+) Tcell memory response specific for the expressed immunogen, antigen orepitope) which is boostable (can be boosted) by a subsequentadministration (boost) of an inactivated vaccine or immunologicalcomposition or a live recombinant vaccine comprising or consistingessentially of a viral vector, such as a live recombinant poxvirus,containing or consisting essentially of and expressing in vivo at leastthe same immunogen(s) or antigen(s) or epitope(s) expressed by the DNAvaccine. The IFN_(γ)+ T cell memory response specific for the expressedWNV immunogen can be shown in a quantitative enzyme-linked immune spot(ELISPOT) assay using peripheral blood mononuclear cells (PBMCs) (LavalF. et al., Vet. Immunol. Immunopathol., 2002, 90(3-4), 191-201).

The “boost” may be administered from about 2 weeks to about 6 monthsafter the “priming”, such as from about 3 to about 8 weeks after thepriming, and advantageously from about 3 to about 6 weeks after thepriming, and more advantageously, about 4 weeks after the priming.

For equines, the priming can be done with a DNA vaccine or immunogenicor immunological composition comprising or consisting essentially of andexpressing in vivo nucleic acid molecule(s) encoding a WNV immunogen,antigen or epitope according to the invention and the boost isadvantageously done with a vaccine or immunogenic or immunologicalcomposition comprising a recombinant live viral vector (e.g. poxvirus,herpesvirus, adenovirus), such as a recombinant fowlpox virus orrecombinant canarypox virus, recombinant EHV-1 or EHV-4, comprising orconsisting essentially of nucleic acid molecule(s) encoding andexpressing in vivo at least one of the same WNV immunogen(s), antigen(s)or epitope(s) as the DNA vaccine or immunogenic or immunologicalcomposition expresses. In another embodiment these priming and boostvaccines or immunological or immunogenic compositions can be adjuvanted,for instance, by DMRIE-DOPE for the priming DNA vaccine or immunologicalor immunogenic composition and by Carbopol® for the boost recombinantvaccine or immunological or immunogenic composition.

The priming may be performed on a young foal that can have maternalantibodies against WNV (against which immunization or vaccination isdirected). Advantageously, the DNA vaccine or immunological orimmunogenic composition is administered to the young foal from foalingup to and including about 16 weeks of age, such as from foaling up toand including about 8 weeks of age, for instance, from foaling up to andincluding about 6 weeks of age, e.g., from foaling up to and includingabout 4 weeks of age.

For felines, the priming can be done with a DNA vaccine or immunogenicor immunological composition according to the invention comprising orconsisting essentially of and expressing in vivo nucleic acidmolecule(s) encoding a WNV immunogen, antigen or epitope and the boostis advantageously done with a vaccine or immunogenic or immunologicalcomposition comprising or consisting essentially a recombinant liveviral vector (e.g. poxvirus, herpesvirus, adenovirus, advantageouslyrecombinant fowlpox virus or recombinant canarypox virus, recombinantFHV, recombinant canine adenovirus), comprising or consistingessentially of nucleic acid molecule(s) encoding and expressing in vivoat least one WNV immunogen, antigen or epitope that is the same as thatexpressed by the DNA vaccine do. In another embodiment these priming andboost vaccines or immunological or immunogenic compositions can beadjuvanted, for instance, by DMRIE-DOPE for the priming DNA vaccine orimmunological or immunogenic composition and by Carbopol® for the boostrecombinant vaccine or immunological or immunogenic composition.

The priming may be performed on a young kitten that can have maternalantibodies against WNV (against which immunization or vaccination isdirected). The DNA vaccine or immunological or immunogenic compositioncan be administered to the young kitten from birth up to and includingabout 12 weeks of age, for instance, from birth up to and includingabout 8 weeks of age, advantageously from birth up to and includingabout 6 weeks of age, e.g., from birth up to and including about 4 weeksof age.

For canines, the priming can be done with a DNA vaccine or immunogenicor immunological composition according to the invention comprising orconsisting essentially of and expressing in vivo nucleic acidmolecule(s) encoding a WNV immunogen, antigen or epitope and the boostis advantageously done with a vaccine or immunogenic or immunologicalcomposition comprising or consisting essentially a recombinant liveviral vector (e.g. poxvirus, herpesvirus, adenovirus, advantageouslyrecombinant fowlpox virus or recombinant canarypox virus, recombinantCHV, recombinant canine adenovirus), comprising or consistingessentially of nucleic acid molecule(s) encoding and expressing in vivoat least one WNV immunogen, antigen or epitope that is the same as thatexpressed by the DNA vaccine do. In another embodiment these priming andboost vaccines or immunological or immunogenic compositions can beadjuvanted, for instance, by DMRIE-DOPE for the priming DNA vaccine orimmunological or immunogenic composition and by Carbopol® for the boostrecombinant vaccine or immunological or immunogenic composition.

The priming may be performed on a young puppy that can have maternalantibodies against WNV (against which immunization or vaccination isdirected). The DNA vaccine or immunological or immunogenic compositioncan be administered to the young puppy from birth up to and includingabout 12 weeks of age, for instance, from birth up to and includingabout 8 weeks of age, advantageously from birth up to and includingabout 6 weeks of age, e.g., from birth up to and including about 4 weeksof age.

For avians, the priming can be done with a DNA vaccine or immunogenic orimmunological composition according to the invention comprising orconsisting essentially of and expressing in vivo nucleic acidmolecule(s) encoding a WNV immunogen, antigen or epitope and the boostis advantageously done with a vaccine or immunogenic or immunologicalcomposition comprising or consisting essentially a recombinant liveviral vector (e.g. poxvirus, herpesvirus, adenovirus, advantageouslyrecombinant fowlpox virus or recombinant canarypox virus, recombinantHVT, recombinant MDV, recombinant avian adenovirus), comprising orconsisting essentially of nucleic acid molecule(s) encoding andexpressing in vivo at least one WNV immunogen, antigen or epitope thatis the same as that expressed by the DNA vaccine do. In anotherembodiment these priming and boost vaccines or immunological orimmunogenic compositions can be adjuvanted, for instance, by DMRIE-DOPEfor the priming DNA vaccine or immunological or immunogenic compositionand by Carbopol® for the boost recombinant vaccine or immunological orimmunogenic composition.

The priming may be performed on a young avian (bird, e.g., chicken) thatcan have maternal antibodies against WNV (against which immunization orvaccination is directed). The DNA vaccine or immunological orimmunogenic composition can be administered to the young avian (bird,such as chicken) from about one day up to and including about 4 weeks ofage, for instance, from one day up to and including about 3 weeks ofage; and, the boost is administered from about 2 to about 8 weeks afterthe priming, advantageously from about 2 weeks to about 4 weeks afterpriming. For the layers, the boost vaccine or immunological orimmunogenic composition may alternatively be administered to about 17weeks of age for hens, to about 25 weeks of age for ducks and to about30 weeks of age for turkey hens. Another administration of the boostvaccine or immunological or immunogenic composition can be done beforeeach laying period.

In an embodiment, the priming DNA vaccine or immunological orimmunogenic composition comprises or consists essentially of a plasmidencoding and expressing prM-M-E polyprotein, such as the plasmid pFC115(example 17), that so encodes and expresses the prM-M-E polyprotein, andthe boost recombinant vaccine or immunological or immunogeniccomposition comprises or consists essentially of a poxvirus such as acanarypox virus, for instance, the recombinant canarypox virus vCP2017(example 18.1). In another embodiment these priming and boost vaccinesor immunological or immunogenic compositions can be adjuvanted: the DNAvaccine or immunological or immunogenic composition containing theplasmid pFC115 can be adjuvanted by DMRIE-DOPE, such as described inexample 20; and the recombinant vaccine or immunological or immunogeniccomposition containing vCP2017 can be adjuvanted by Carbopol®, such asdescribed in example 19.

In a further embodiment, the priming DNA vaccine or immunological orimmunogenic composition comprises or consists essentially of a plasmidencoding and expressing prM-M-E polyprotein, such as the plasmid pFC115(example 17) and the boost recombinant vaccine or immunological orimmunogenic composition comprises a poxvirus such as a fowlpox virus,e.g., the recombinant fowlpox virus vFP2000 (example 28). In anotherembodiment these priming and boost vaccines or immunological orimmunogenic compositions can be adjuvanted: the DNA vaccine orimmunological or immunogenic composition containing the plasmid pFC115can be adjuvanted by DMRIE-DOPE, as described in example 20; and therecombinant vaccine or immunological or immunogenic compositioncontaining vFP2000 can be adjuvanted by Carbopol®, as described inexample 29.

The invention also relates to kits for performing prime-boost methodscomprising or consisting essentially of a priming vaccine orimmunological or immunogenic composition and a boost vaccine orimmunological or immunogenic compositions in separate containers,optionally with instructions for admixture and/or administration.

The amounts (doses) administered in the priming and the boost and theroute of administration for the priming and boost can be as hereindiscussed, such that from this disclosure and the knowledge in the art,the prime-boost regimen can be practiced without undue experimentation.Furthermore, from the disclosure herein and the knowledge in the art,the skilled artisan can practice the methods, kits, etc. herein withrespect to any of the herein-mentioned target species.

These methods can comprise, consist essentially of or consist of theadministration of an effective quantity of an immunogenic composition orvaccine according to the invention. This administration can be by theparenteral route, e.g. by subcutaneous, intradermic or intramuscularadministration, and/or by oral and/or nasal routes. Advantageously, thisadministration is intramuscularly or subcutaneously. One or moreadministrations can take place, such as two administrations.

Vaccines or immunogenic compositions can be injected by a needleless,liquid jet injector or powder jet injector. For plasmids it is alsopossible to use gold particles coated with plasmid and ejected in such away as to penetrate the cells of the skin of the subject to be immunized(Tang et al., Nature 1992, 356, 152-154). Other documents cited andincorporated herein may be consulted for administration methods andapparatus of vaccines or immunogenic compositions of the invention. Theneedleless injector can also be for example Biojector 2000 (BiojectInc., Portland Oreg., USA).

Advantageously, the immunogenic compositions and vaccines according tothe invention comprise or consist essentially of or consist of aneffective quantity to elicit an immunological response and/or aprotective immunological response of one or more expression vectorsand/or polypeptides as discussed herein; and, an effective quantity canbe determined from this disclosure, including the documents incorporatedherein, and the knowledge in the art, without undue experimentation.

In the case of immunogenic compositions or vaccines based on a plasmidvector, a dose can comprise, consist essentially of or consist of, ingeneral terms, about in 10 μg to about 2000 μg, advantageously about 50μg to about 1000 μg. The dose volumes can be between about 0.1 and about2 ml, preferably between about 0.2 and about 1 ml.

These doses and dose volumes are suitable for the vaccination of equinesand other target species that are mammals such as canines, felines.

For the vaccination or immunization of an avian, a dose isadvantageously between about 10 μg and about 500 μg and preferablybetween about 50 μg and about 200 μg. The dose volumes can be betweenabout 0.1 and about 1 ml, preferably between about 0.2 and about 0.5 ml.

One skilled in the art can determine the effective plasmid dose to beused for each immunization or vaccination protocol and species from thisdisclosure and the knowledge in the art.

In the case of immunogenic compositions or vaccines based on a poxvirus,a dose can be between about 10² pfu and about 10⁹ pfu.

For equines and other target species that are mammals such as felinesand canines, when the vector is a vaccinia virus, the dose is moreadvantageously between about 10⁴ pfu and about 10⁹ pfu, preferablybetween about 10⁶ pfu and about 10⁸ pfu and when the vector is acanarypox virus, the dose is more advantageously between about 10⁵ pfuand about 10⁹ pfu and preferably between about 10^(5.5) pfu or about 10⁶pfu and about 10⁸ pfu.

For an avian, when the vector is a poxvirus such as a canarypox virus,the dose is more advantageously between about 10³ pfu and about 10⁷ pfu,preferably between about 10⁴ pfu and about 10⁶ pfu; and, when the vectoris a poxvirus such as a fowlpox virus, the dose is more advantageouslybetween about 10² pfu and about 10⁵ pfu, preferably between about 10³pfu and about 10⁵ pfu. From this disclosure and the knowledge in theart, the skilled artisan can determine the suitable dose when the vectoris another avipox virus, such as a dovepox, pigeonpox, etc.

In the case of immunogenic compositions or vaccines for a mammaliantarget species, based on a viral vector other than a poxvirus, such as aherpes viruses or adenovirus, a dose is generally between about 10³ pfuand about 10⁸ pfu; and, in the case of suchnon-poxvirus-viral-vector-based immunogenic compositions for avianspecies or avian vaccines, a dose is generally between about 10³ pfu andabout 10⁶ pfu. For such non-poxvirus-viral-vector-based immunogenic orvaccine compositions for larger target mammal species, e.g., larger cats(e.g., kept in a zoo) or equines, e.g., in the case of equineimmunogenic or vaccine compositions, a dose is advantageously betweenabout 10⁶ pfu and about 10⁸ pfu.

The dose volume of immunogenic and vaccine compositions for targetspecies that are mammals, e.g., the dose volume of equine immunogenic orvaccine compositions, based on viral vectors, e.g.,non-poxvirus-viral-vector-based immunogenic or vaccine compositions, isgenerally between about 0.5 and about 2.5 ml, such as between about 0.5and about 2.0 ml, preferably between about 1.0 and about 2.0 ml,preferably about 1.0 ml. The dose volume of immunogenic or vaccinecompositions for avians based on viral vectors, e.g., the dose volume ofnon-poxvirus-viral-vector-based avian immunogenic or vaccinecompositions, is generally between about 0.1 and about 1.0 ml,preferably between about 0.1 and about 0.5 ml and more advantageouslybetween about 0.2 and about 0.3 ml. Also in connection with such avaccine or immunogenic composition, from the disclosure herein and theknowledge in the art, the skilled artisan can determine the number ofadministrations, the administration route, and the doses to be used foreach immunization or vaccination protocol, without any undueexperimentation. For instance, there can be two administrations to ahorse, e.g. at 35 day intervals.

In the case of subunit immunogenic compositions or subunit vaccines,with reference to the amount of active ingredient, e.g., subunit(antigen, immunogen, epitope) a dose comprises or consists essentiallyof or consists of, in general terms, about 10 μg to about 2000 μg,advantageously about 50 μg to approximately 1000 μg. The dose volume ofsuch immunogenic or vaccine compositions for target species that aremammals, e.g., for equines, is generally between about 1.0 and about 2.0ml, preferably between about 0.5 and about 2.0 ml and moreadvantageously about 1.0 ml. The dose volumes of such immunogenic orvaccine compositions avians is generally between about 0.1 and about 1.0ml, preferably between about 0.1 and about 0.5 ml, and moreadvantageously between 0.2 and 0.3 ml. Also for such a vaccine orimmunogenic composition, the skilled artisan, from this disclosure andthe knowledge in the art, can, without any undue experimentation,determine the number of administrations, the administration route andthe doses to be used for each immunization or vaccination protocol.

The invention also relates to the use of an in vivo expression vector ora preparation of vectors and/or polypeptides according to the invention,for the formulation of an immunogenic composition or a vaccine intendedto protect a target species, or elicit in the target species animmunological response, against the WN virus, and in certainembodiments, against at least one other pathogenic agent.

A vaccine based on plasmid or a viral vaccine expressing one or moreproteins of the WN virus or a WN subunit vaccine according to thepresent invention will not induce in the immunized or vaccinated animalantibodies against other proteins of the virus, which are not presentedin or by the immunogenic composition or vaccine (e.g., not present inthe immunogenic composition or vaccine and/or not expressed by theimmunogenic composition or vaccine). By this feature, the instantinvention provides differential diagnostic methods. The presentinvention makes it possible to make a distinction between animalsinfected by the WN pathogenic virus and animals vaccinated or immunizedwith vaccines or compositions according to the invention. In the former,proteins and/or antibodies directed against them are present and can bedetected by an antigen-antibody reaction. In the latter (the animalsvaccinated or immunized according to the invention), this is not thecase, as such animals remain negative in such an antigen-antibodyreaction as to proteins not presented in or by the immunogenic orvaccine composition or antibodies thereto. In order to bring about thisdiscrimination, the diagnostic method employs a protein which is notrepresented in or by the vaccine or immunogenic composition (not presentand/or not expressed), e.g. protein C or protein NS1, NS2A, NS2B or NS3when it is not represented in the vaccine or immunogenic composition.

Accordingly, the instant invention comprehends diagnostic assays or kitsthat employ a protein or antibody thereto that is not presented in or bya vaccine or immunogenic composition of the invention; and, kits thatcontain such a diagnostic assay or kit and such a vaccine or immunogeniccomposition, whereby the user can innoculate and/or vaccinate animalsand thereafter test the animals, to determine those animals that havebeen exposed to WNV vs. those animals that have only been immunizedand/or vaccinated against WNV.

Thus, the present invention relates to the use of vectors, preparationsand polypeptides according to the invention for the preparation ofimmunogenic compositions and vaccines making it possible to discriminatebetween vaccinated or immunized animals and infected animals.

The instant invention also relates to an immunization and vaccinationmethod associated with a diagnostic method permitting such adiscrimination.

The protein selected for the diagnosis or one of its fragments orepitopes is used as the antigen in the diagnostic test and/or is usedfor producing polyclonal or monoclonal antibodies.

The one skilled in the art has sufficient practical knowledge to producethese antibodies and to implement antigens and/or antibodies inconventional diagnostic methods, e.g. ELISA tests, and thereby performdifferential diagnostic tests according to the instant invention.

The invention will now be further described and illustrated by way ofthe following, non-limiting examples.

EXAMPLES

All the constructions are implemented using standard molecular biologymethods (cloning, digestion by restriction enzymes, synthesis of acomplementary single-strand DNA, polymerase chain reaction, elongationof an oligonucleotide by DNA polymerase, etc.) described by Sambrook J.et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold SpringHarbor Laboratory, Cold Spring Harbor. N.Y., 1989). All the restrictionfragments used for these examples of the present invention, as well asthe various polymerase chain reaction (PCR) products are isolated andpurified using the Qiagen gel extraction or PCR purification kits

Example 1 Culture of the West Nile Fever Virus

For amplification, West Nile fever virus NY99 (Lanciotti R. S. et al.,Science, 1999, 286, 2333-7)) are cultured on VERO cells (monkey renalcells), obtainable from the American Type Culture Collection (ATCC)under no. CCL-81.

The VERO cells were cultured in 25 cm² Falcon with eagle-MEM mediumsupplemented by 1% yeast extracts and 10% calf serum containingapproximately 100,000 cells/ml. The cells were cultured at +37° C. undera 5% CO₂ atmosphere.

After three days the cellular layer reaches to confluence. The culturemedium was then replaced by the eagle-MEM medium supplemented by 1%yeast extract and 0.1% cattle serum albumin and the West Nile fevervirus was added at a rate of 5 pfu/cell.

When the cytopathogenic effect (CPE) was complete (generally 48 to 72hours after the start of culturing), the viral suspensions wereharvested and then clarified by centrifugation and frozen at −70° C. Ingeneral, three to four successive passages were necessary for producinga viral batch, which is stored at −70° C.

Example 1.1 Construction of an Insertion Plasmid for the Canarypox C5Locus

FIG. 13 (SEQ ID NO:77) is the sequence of a 5 kb segment of canarypoxDNA, encoding an ORF designated C5 initiating at position 1864 andterminating at position 2187. The following describes a C5 insertionplasmid constructed by deleting the majority of the C5 ORF and replacingit with the Virogenetics VQ marker, the H6 promoter, a multiple cloningsite (MCS) and transcriptional and translational termination sequencesin all reading rames. A 1590 by PCR fragment, containing the upstreamC5R arm is amplified from genomic canarypox DNA using primers C5A1 (SEQID NO:76) and C5B1 (SEQ ID NO:68). This fragment includes an EcoR I siteat the 5′-end, termination sequences and an MCS containing BamH I, Cla Iand Xma I sites at the 3′-end. A 458 by PCR fragment, containing thedownstream C5L arm is amplified from genomic canarypox DNA using primersC5C1 (SEQ ID NO:69) and C5D1 (SEQ ID NO:70). The fragment includes 5′BamH I, Cla I and Xma I sites, termination sequences and a Pst I site atthe 3′-end. The PCR fragments were fused together by re-amplifying withprimers C5A and CSD, generating a 2030 by EcoR I-Pst I fragment, whichis cloned into pUC 8, generating pUC/C5L/B Cla Xm/C5R. Oligonucleotides(SEQ ID NO:71) were used to introduce a unique Not I sequence at the5′-end of the C5R arm, by inserting into the EcoR I site, generatingpUC/Not I/C5R/MCS/C5L.

The Virogenetics VQ marker is contained on plasmid pRW823 and thevaccinia H6 promoter is contained on plasmid pBSH6-1. An 82 by fragmentcontaining the VQ marker and a 5′ BamH I site, was PCR amplified frompRW823 using primers VQA1 (SEQ ID NO:72) and VQB1 (SEQ ID NO:73). A 176by fragment containing the H6 promoter and recognition sequences for amultiple cloning site containing Asp718 I, Xho I, Xba I, Cla I and SmaI, was amplified using primers H6A1 (SEQ ID NO:74) and H6B1 (SEQ IDNO:75). The VQ and H6 fragments were pooled and re-amplified usingprimers VQA1 and H6B1 to generate a 232 by VQ/H6p/MCS fragment (FIG. 14,SEQ ID NO:82) that was inserted into pUC/C5L/B Cla Xm/C5R between theBamH I and Xma I sites. FIG. 15 shows the resultant plasmid,pNVQH6C5LSP-18, a C5 insertion plasmid containing the H6 promoter,transcription and translation terminators functional in all readingframes, and a MCS.

Sequences of the PCR primers and oligonucleotides: (SEQ ID NO: 76)Primer C5A1 5′ GGCCGAATTCTGAATGTTAAATGTTATACTTT 3′ (SEQ ID NO: 68)Primer C5B1 5′ CCCGGGATCGATGGATCCTTTTTATAGCTAATTAGTCACGTACCTTTGAGAGTACCACTTCAGCTA 3′ (SEQ ID NO: 69) Primer C5C15′ GGATCCATCGATCCCGGGTTTTTATGACTAGTTAATCACGGCCGCTT ATAAAGATCTAAAATGCAT3′ (SEQ ID NO: 70) Primer C5D1 5′ GGCTGCAGGTATTCTAAACTAGGAATAGAT 3′ (SEQID NO: 71) Oligonucleotide for Not I 5′ AATTGCGGCCGC 3′ (SEQ ID NO: 72)Primer VQA1 5′ AAAGGATCCGGGTTAATTAATTAGTCATC 3′ (SEQ ID NO: 73) PrimerVQB1 5′ AATAAAGAAGCTCTAATTAATTAACGAGCAGATA 3′ (SEQ ID NO: 74) PrimerH6A1 5′ TCGTTAATTAATTAGAGCTTCTTTATTCTATACTTAAAAAG 3′ (SEQ ID NO: 75)Primer H6B1 5′ AAAACCCGGGATCGATTCTAGACTCGAGGGTACCTACGATACAAACT TAACGGATA3′

Example 2 Extraction of Viral RNA from the West Nile Fever Virus

The viral RNA contained in 100 ml of viral suspension of the West Nilefever virus strain NY99 was extracted after thawing with solutions ofthe High Pure Viral RNA Kit Cat #1 858 882, Roche MolecularBiochemicals, whilst following the instructions of the supplier for theextraction stages. The RNA sediment obtained at the end of extractionwas resuspended with 1 to 2 ml of RNase-free, sterile distilled water.

Example 3 Construction of Plasmid pFC101

The complementary DNA (cDNA) of the West Nile fever virus NY99 wassynthesized with the Gene Amp RNA PCR Kit (Cat # N 808 0017,Perkin-Elmer, Norwalk, Conn. 06859, USA) using the conditions suppliedby the manufacture.

A reverse transcriptase polymerase chain reaction (RT-PCR reaction) wascarried out with 50 μl of viral RNA suspension of the West Nile fevervirus NY99 (Example 2) and with the following oligonucleotides:

(SEQ ID NO: 1) FC101 (30 mer) 5′TTTTTTGAATTCGTTACCCTCTCTAACTTC 3′ and(SEQ ID NO: 2) FC102 (33 mer) 5′TTTTTTTCTAGATTACCTCCGACTGCGTCTTGA 3′

This pair of oligonucleotides allows the incorporation of an EcoRIrestriction site, a XbaI restriction site and a stop codon at 3′ of theinsert.

The synthesis of the first cDNA strand takes place by elongation ofoligonucleotide FC102, following the hybridization of the latter withthe RNA matrix.

The synthesis conditions of the first cDNA strand were a temperature of42° C. for 15 min, then 99° C. for 5 min and finally 4° C. for 5 min.The conditions of the PCR reaction in the presence of the pair ofoligonucleotides FC101 and FC102 were a temperature of 95° C. for 2 min,then 35 cycles (95° C. for 1 min, then 62° C. for 1 min and 72° C. for 2min) and finally 72° C. for 7 min to produce a 302 by fragment.

This fragment was digested by EcoRI and then by XbaI in order toisolate, following agarose gel electrophoresis, the approximately 290 byEcoRI-XbaI fragment, which was called fragment A.

The pVR1020 eukaryotic expression plasmid (C. J. Luke et al. ofInfectious Diseases, 1997, 175, 95-97) derived from the plasmid pVR1012(FIG. 1 and example 7 of WO98/03199-Hartikka J. et al., 1997, Human GeneTherapy, 7, 1205-1217), contains the frame encoding the signal sequenceof the human tissue plasminogen activator (tPA).

A pVR1020 plasmid was modified by BamHI-BglII digestion and insertion ofa sequence containing several cloning sites (BamHI, NotI, EcoRI, XbaI,PmlI, PstI, BglII) resulting from hybridization of the followingoligonucleotides.

(SEQ ID NO: 3) BP326 (40 mer) 5′GATCTGCAGCACGTGTCTAGAGGATATCGAATTCGCGGCC3′ and (SEQ ID NO: 4) BP329 (40 mer)5′GATCCGCGGCCGCGAATTCGATATCCTCTAGACACGTGCT 3′

The thus obtained vector with a size of approximately 5105 base pairs(or bp) was called pAB110.

Fragment A was ligated with the pAB110 expression plasmid previouslydigested by XbaI and EcoRI, in order to give the plasmid pFC101 (5376bp). Under the control of the early promoter of human cytomegalovirus orhCMV-IE (human Cytomegalovirus Immediate Early), the plasmid contains aninsert encoding the signal sequence of the activator of tPA followed bythe sequence encoding the protein prM.

Example 3.1 Construction of a DNA Immunization Vector, pVR1012WNVprM-M-E, pSL-5448-1-1

The construction scheme is shown in FIG. 7.

Plasmid pTriEx-WNV containing the NY99 WNV prM-M-E genes, was receivedfrom Cornell University. There is a poly-His tag at the 3′ end of the Egene. A 1.2 kb Cla I-Xba I 3′-WNV fragment was PCR amplified usingprimers 7601.SL and 7617.SL, to remove the poly-His tag and introduce astop codon and Xba I site for cloning. The resultant fragment was clonedinto pCR 2.1, generating pDS-2905-3-1.

Plasmid pVR1012 is a DNA immunization vector containing the human CMVpromoter, intronA, a multiple cloning site, and a kanamycin resistancegene and has been described by Hartikka et al (Human Gene Therapy7:1205-1217, 1997). The pVR1012 vector was digested with EcoR V and XbaI and ligated with the 1.2 kb Cla I-Xba I 3′-insert from pDS-2905-3-1and a 0.7 kb EcoR V-Cla I 5′-fragment from pTri-Ex-WNV, to generatepDS-2933-2-2.

In order to introduce a 5′ Kozak sequence, the Pst I-EcoR V fragment ofpDS-2933-2-2 was replaced by annealed oligonucleotides 7743.SL and7744.SL, generating clone pSL-5448-1-1, pVR1012 prM-M-E. The sequence ofthe WNV prM-M-E region is shown in FIG. 8.

Example 3.2 Analysis of the Immunogenicity of pSL-5448-1-1, pVR1012 WNVprM-M-E in Mice

Six to eight week old BALB/c mice were immunized intramuscularly with100 μg of plasmid DNA in PBS, on days 0, 14, 28, and 42. Bleeds weretaken on days 0, 28, 42 and 56, processed and analysed by immunoblot.

Samples for immunoblot analysis were prepared by transient transfectionof Chinese Hamster Ovary (CHO) cells with pTriEx-WNV DNA. CHO cells weretransfected with 10 μg of pTriEx-WNV DNA using electroporation atsettings 1.5 kV, 25 uF and infinite resistance. Mock samples wereprepared by the electroporation of CHO cells at the same settings,without DNA. After approximately 65 hours, supernatants were harvestedand clarified by spinning at 3000 rpm for 5 min. The plates were washedtwice with PBS, then 500 μl of PBS was added and the cells were scrapedoff. After spinning at 3000 rpm for 5 min, the supernatant was removedand the cells resuspended in 100 μl of SDS-PAGE lysis buffer.

Immunoblots were performed using the CHO/pTriEx-WNV and CHO/mock pelletsto assess antigenicity and specificity of the mouse antisera. Sampleswere suspended in SDS-PAGE loading buffer minus β-mercaptoethanol andseparated on a 12% SDS-PAGE gel before electrotransfer to immobilon Pnylon membrane. The membranes were processed and probed with 1:1000dilution of mouse anti-WNV antisera. Peroxidase-conjugated goatanti-mouse antisera was used as secondary antibody and bands werevisualized using luminol reagents (NEN). All five mouse anti-WNVantisera reacted specifically with a single protein band in thepTriEx-WNV samples, of the expected size for the WNV E protein. None ofthe five antisera reacted with anything in the mock samples.

The serum samples were also assayed for virus neutralizing antibodiesand titres were found to range between 1:8 and 1:128.

Example 4 Construction of Plasmid pFC102

The complementary DNA (cDNA) of the West Nile fever virus NY99 wassynthesized with the Gene Amp RNA PCR Kit (Cat # N 808 0017,Perkin-Elmer, Norwalk, Conn. 06859, USA) using the conditions providedby the supplier.

A reverse transcriptase polymerase chain reaction (RT-PCR reaction) wascarried out with 50 μl of viral RNA suspension of the West Nile fevervirus NY99 (Example 2) and with the following oligonucleotides:

(SEQ ID NO: 5) FC103 (30 mer) 5′TTTTTTGAATTCTCACTGACAGTGCAGACA 3′ and(SEQ ID NO: 6) FC104 (33 mer) 5′TTTTTTTCTAGATTAGCTGTAAGCTGGGGCCAC 3′

This pair of oligonucleotides allows the incorporation of an EcoRIrestriction site and a XbaI restriction site and a stop codon at 3′ ofthe insert.

The first cDNA strand was synthesized by elongation of oligonucleotideFC104, following the hybridization of the latter on the RNA matrix.

The synthesis conditions of the first cDNA strand were a temperature of42° C. for 15 min, then 99° C. for 5 min and finally 4° C. for 5 min.The conditions of the PCR reaction in the presence of the pair ofoligonucleotides FC103 and FC104 were a temperature of 95° C. for 2 min,then 35 cycles (95° C. for 1 min, then 62° C. for 1 min and 72° C. for 2min) and finally 72° C. for 7 min to produce a 252 by fragment.

This fragment was digested by EcoRI and then XbaI in order to isolate,following agarose gel electrophoresis, the approximately 240 byEcoRI-XbaI fragment. This fragment was ligated with the pAB110expression plasmid (Example 3) previously digested by XbaI and EcoRI inorder to give the plasmid pFC102 (5326 bp). Under the control of theearly human cytomegalovirus or hCMV-IE (human Cytomegalovirus ImmediateEarly) promoter, this plasmid contains an insert encoding the signalsequence of the activator of tPA, followed by the sequence encoding theprotein M.

Example 5 Construction of Plasmid pFC103

The complementary DNA (cDNA) of the West Nile fever virus NY99 wassynthesized with the Gene Amp RNA PCR Kit (Cat # N 808 0017,Perkin-Elmer, Norwalk, Conn. 06859, USA) using the conditions providedby the supplier.

A reverse transcriptase polymerase chain reaction (RT-PCR reaction) wascarried out with 50 μl of viral RNA suspension of the West Nile fevervirus NY99 (Example 2) and with the following oligonucleotides:

(SEQ ID NO: 7) FC105 (30 mer) 5′TTTTTTGAATTCTTCAACTGCCTTGGAATG 3′ and(SEQ ID NO: 8) FC106 (33 mer) 5′TTTTTTTCTAGATTAAGCGTGCACGTTCACGGA 3′.

This pair of oligonucleotides allows the incorporation of an EcoRIrestriction site and a XbaI restriction site, together with a stop codonat 3′ of the insert.

The synthesis of the first cDNA strand takes place by elongation ofoligonucleotide FC106, following its hybridization with the RNA matrix.

The synthesis conditions of the first cDNA strand were a temperature of42° C. for 15 min, then 99° C. for 5 min and finally 4° C. for 5 min.The PCR reaction conditions in the presence of the pair ofoligonucleotides FC105 and FC106 were a temperature of 95° C. for 2 min,then 35 cycles (95° C. for 1 min, then 62° C. for 1 min and 72° C. for 2min), and finally 72° C. for 7 min for producing a 1530 by fragment.

This fragment was digested by EcoRI and then by XbaI in order toisolate, following agarose gel electrophoresis, the approximately 1518by EcoRI-XbaI fragment. This fragment was ligated with the pAB110expression plasmid (Example 3) previously digested by XbaI and EcoRI inorder to give the plasmid pFC103 (6604 bp). Under the control of theearly promoter of human cytomegalovirus or hCMV-IE (humanCytomegalovirus Immediate Early), the plasmid contains an insertencoding the signal sequence of the activator of tPA, followed by thesequence encoding the protein E.

Example 6 Construction of Plasmid pFC104

The complementary DNA (cDNA) of the West Nile fever virus NY99 wassynthesized with the Gene Amp RNA PCR Kit (Cat # N 808 0017,Perkin-Elmer, Norwalk, Conn. 06859, USA) using the conditions providedby the supplier.

A reverse transcriptase polymerase chain reaction (RT-PCR reaction) wascarried out with 50 μl of viral RNA suspension of the West Nile fevervirus NY99 (Example 2) and with the following oligonucleotides:

FC101 (30 mer) (SEQ ID NO: 1) and FC106 (33 mer) (SEQ ID NO: 8)

This pair of oligonucleotides allows the incorporation of an EcoRIrestriction site, a XbaI restriction site and a stop codon at 3′ of theinsert.

Synthesis of the first cDNA strand takes place by elongation ofoligonucleotide FC106, following its hybridization with the RNA matrix.

The synthesis conditions of the first cDNA strand were a temperature of42° C. for 15 min, then 99° C. for 5 min and finally 4° C. for 5 min.The PCR reaction conditions in the presence of the pair ofoligonucleotides FC101 and FC106 are a temperature of 95° C. for 2 min,then 35 cycles (95° C. for 1 min, then 62° C. for 1 min and 72° C. for 2min) and finally 72° C. for 7 min in order to produce a 2031 byfragment.

This fragment was digested by EcoRI and then XbaI in order to isolate,following agarose gel electrophoresis, the approximately 2019 byEcoRI-XbaI fragment. This fragment was ligated with the pAB110expression plasmid (Example 3), previously digested by XbaI and EcoRI inorder to give the pFC104 plasmid (7105 bp). Under the control of theearly human cytomegalovirus promoter or hCMV-IE (human CytomegalovirusImmediate Early), the plasmid contains an insert encoding the signalsequence of the activator of tPA, followed by the sequence encoding theprotein prM-M-E.

Example 7 Construction of plasmid pFC105

The complementary DNA (cDNA) of the West Nile fever virus NY99 wassynthesized with the Gene Amp RNA PCR Kit (Cat # N 808 0017,Perkin-Elmer, Norwalk, Conn. 06859, USA) using the conditions providedby the supplier.

A reverse transcriptase polymerase chain reaction (RT-PCR reaction) wascarried out with 50 μl of viral RNA suspension of the West Nile fevervirus NY99 (Example 2) and with the following oligonucleotides:

(SEQ ID NO: 9) FC107 (36 mer) 5′TTTTTTGATATCACCGGAATTGCAGTCATGATTGGC 3′and (SEQ ID NO: 8) FC106 (33 mer).

This pair of oligonucleotides allows the incorporation of an EcoRVrestriction site, a XbaI restriction site and a stop codon at 3′ of theinsert.

Synthesis of the first cDNA strand takes place by elongation of theFC106 oligonucleotide, following its hybridization with the RNA matrix.

The synthesis conditions of the first cDNA strand were a temperature of42° C. for 15 min, then 99° C. for 5 min and finally 4° C. for 5 min.The PCR reaction conditions in the presence of the pair ofoligonucleotides FC106 and FC107 are a temperature of 95° C. for 2 min,then 35 cycles (95° C. for 1 min, then 62° C. for 1 min and 72° C. for 2min) and finally 72° C. for 7 min in order to produce a 2076 byfragment.

This fragment was digested by EcoRV and then XbaI in order to isolate,following agarose gel electrophoresis, the approximately 2058 byEcoRV-XbaI fragment.

This fragment was ligated with the pVR1012 expression plasmid,previously digested by XbaI and EcoRV, in order to give the plasmidpFC105 (6953 bp). Under the control of the early human cytomegaloviruspromoter or hCMV-IE (human Cytomegalovirus Immediate Early), thisplasmid contains an insert encoding the polyprotein prM-M-E.

Example 8 Construction of Plasmid pFC106

The complementary DNA (cDNA) of the West Nile fever virus NY99 wassynthesized with the Gene Amp RNA PCR Kit (Cat # N 808 0017,Perkin-Elmer, Norwalk, Conn. 06859, USA) using the conditions providedby the supplier.

A reverse transcriptase polymerase chain reaction (RT-PCR reaction) wascarried out with 50 μl of viral RNA suspension of the West Nile fevervirus NY99 (example 2) and with the following oligonucleotides:

FC108 (36 mer) (SEQ ID NO: 10) 5′TTTTTTGATATCATGTATAATGCTGATATGATTGAC 3′and FC109 (36 mer) (SEQ ID NO: 11)5′TTTTTTTCTAGATTAACGTTTTCCCGAGGCGAAGTC 3′

This pair of oligonucleotides allows the incorporation of an EcoRVrestriction site, a XbaI restriction site, an initiating ATG codon in 5′and a stop codon at 3′ of the insert.

Synthesis of the first cDNA strand takes place by elongation of theoligonucleotide FC109, following its hybridization with the RNA matrix.

The synthesis conditions of the first cDNA strand were a temperature of42° C. for 15 min, then 99° C. for 5 min and finally 4° C. for 5 min.The PCR reaction conditions in the presence of the pair of nucleotidesFC108 and FC109 are a temperature of 95° C. for 2 min, then 35 cycles(95° C. for 1 min, 62° C. for 1 min and then 72° C. for 2 min) andfinally 72° C. for 7 min to produce a 2973 by fragment.

This fragment was digested by EcoRV and then XbaI in order to isolate,following agarose gel electrophoresis, the approximately 2955 byEcoRV-XbaI fragment.

This fragment was ligated with the pVR 1012 expression plasmidpreviously digested by XbaI and EcoRV in order to give the plasmidpFC106 (7850 bp). Under the control of the early human cytomegaloviruspromoter or hCMV-IE (human Cytomegalovirus Immediate Early), thisplasmid contains an insert encoding the polyprotein NS2A-NS2B-NS3.

Example 9 Construction of Donor Plasmid for Insertion into C5 Site ofCanarypox Virus (ALVAC)

FIG. 16 of U.S. Pat. No. 5,756,103 shows the sequence of a genomic DNA3199 by fragment of the canarypox virus. Analysis of this sequence hasrevealed an open reading frame (ORF) called C5.H, which starts atposition 1538 and ends at position 1859. The construction of aninsertion plasmid leading to the deletion of the ORF C5.H and itsreplacement by a multiple cloning site flanked by transcription andtranslation stop signals was implemented in the following way.

A PCR reaction was performed on the basis of the matrix constituted bygenomic DNA of the canarypox virus and with the followingoligonucleotides:

C5A1 (42 mer) (SEQ ID NO: 12):5′ATCATCGAGCTCCAGCTGTAATTCATGGTCGAAAAGAAGTGC 3′ and C5B1 (73 mer) (SEQID NO: 13): 5′GAATTCCTCGAGCTGCAGCCCGGGTTTTTATAGCTAATTAGTCATTTTTTGAGAGTACCACTTCAGCTACCTC 3′in order to isolate a 223 by PCR fragment (fragment B).

A PCR reaction was carried out on the basis of the matrix constituted bygenomic DNA of the canarypox virus and with the followingoligonucleotides:

C5C1 (72 mer) (SEQ ID NO: 14):5′CCCGGGCTGCAGCTCGAGGAATTCTTTTTATTGATTAACTAGTCATTATAAAGATCTAAAATGCATAATTTC 3′ and C5D1 (45 mer) (SEQ ID NO: 15):5′GATGATGGTACCGTAAACAAATATAATGAAAAGTATTCTAAACTA 3′in order to isolate a 482 by PCR fragment (fragment C).

Fragments B and C were hybridized together in order to serve as a matrixfor a PCR reaction performed with the oligonucleotides C5A1 (SEQ ID NO:12) and C5D1 (SEQ ID NO: 15) in order to generate a 681 by PCR fragment.This fragment was digested by the restriction enzymes SacI and KpnI inorder to isolate, following agarose gel electrophoresis, a 664 bySacI-KpnI fragment. This fragment was ligated with the pBlueScript® IISK+ vector (Stratagene, La Jolla, USA, Cat #212205), previously digestedby the restriction enzymes SacI and KpnI, in order to give the plasmidpC5.H. The sequence of this plasmid was verified by sequencing. Thisplasmid contains 166 by of sequences upstream of ORF C5.H (left flankingarm C5L.H), an early transcription stop signal, stop codons in 6 readingframes, a multiple cloning site containing restriction sites SmaI, PstI,XhoI and EcoRI and finally 425 by of sequences located downstream of ORFC5.H (right flanking arm C5R.H).

The plasmid pMP528HRH (Perkus M. et al. J. Virol. 1989, 63, 3829-3836)was used as the matrix for amplifying the complete sequence of thevaccinia promoter H6 (GenBank access no. M28351) with the followingoligonucleotides:

JCA291 (34 mer) (SEQ ID NO: 16) 5′AAACCCGGGTTCTTTATTCTATACTTAAAAAGTG 3′and JCA292 (43 mer) (SEQ ID NO: 17)5′AAAAGAATTCGTCGACTACGATACAAACTTAACGGATATCGCG 3′in order to amplify a 149 by PCR fragment. This fragment was digested byrestriction enzymes SmaI and EcoRI in order to isolate, followingagarose gel electrophoresis, a 138 by SmaI-EcoRI restriction fragment.This fragment was then ligated with the plasmid pC5, previously digestedby SmaI and EcoRI, in order to give the plasmid pFC107.

Example 10 Construction of the Recombinant Virus vCP1712

A PCR reaction was performed using the plasmid pFC105 (example 7) as thematrix and the following oligonucleotides:

FC110 (33 mer (SEQ ID NO: 18): 5′TTTTCGCGAACCGGAATTGCAGTCATGATTGGC 3′and FC111 (39 mer) (SEQ ID NO: 19):5′TTTTGTCGACGCGGCCGCTTAAGCGTGCACGTTCACGGA 3′in order to amplify an approximately 2079 by PCR fragment. This fragmentwas digested by restriction enzymes NruI and SalI in order to isolate,following agarose gel electrophoresis, an approximately 2068 byNruI-SalI restriction fragment. This fragment was then ligated withplasmid pFC107 (example 9) previously digested by restriction enzymesNruI and SalI in order to give the plasmid pFC108, which containsC5L-H6p-WNV prM-M-E-C5R.

Plasmid pFC108 was linearized by NotI, then transfected in primarychicken embryo cells. The cells were then infected with the canarypoxvirus (ALVAC strain) according to the previously described calciumphosphate precipitation method (Panicali and Paoletti, Proc. Nat. Acad.Sci. 1982, 79, 4927-4931; Piccini et al. In Methods in Enzymology, 1987,153, 545-563, publishers Wu R. and Grossman L. Academic Press). Positiveplaques were selected on the basis of a hybridization with aradioactively labelled probe specific to the nucleotide sequence of theenvelope glycoprotein E. These plaques underwent 2-4 successiveselection/purification cycles until a pure population was isolated. Arepresentative plaque corresponding to in vitro recombination betweenthe donor plasmid pFC108 and the genome of the ALVAC canarypox virus wasthen amplified and the recombinant virus stock obtained was designatedvCP1712. (The actual vCP2017, which contains the full-length promoterand signal sequence, was derived after two rounds of screening).

Example 11 Construction of the Recombinant Virus vCP1713

Plasmid pFC104 (Example 6) was digested by the restriction enzyme SalIand PmlI in order to isolate, following agarose gel electrophoresis, anapproximately 2213 by PmlI-SalI restriction fragment. This fragment wasligated with plasmid pFC107 (Example 9) previously digested by the NruIand SalI restriction enzymes in order to give the plasmid pFC109.

Plasmid pFC109 was linearized by NotI, then transfected in primarychicken embryo cells infected with the canarypox virus (ALVAC strain)according to the method of Example 10. A representative plaquecorresponding to in vitro recombination between the donor plasmid pFC109and the genome of the ALVAC canarypox virus was selected on the basis ofa hybridization of a radioactively labelled probe specific to thenucleotide sequence of the envelope glycoprotein E and was thenamplified. The recombinant virus stock obtained was designated vCP1713,ALVAC WNV prM-M-E.

Example 12 Construction of the Recombinant Virus vCP1714

Plasmid pFC103 (Example 5) was digested by the SalI and PmlI restrictionenzymes in order to isolate, following agarose gel electrophoresis, anapproximately 1712 by PmlI-SalI restriction fragment. This fragment wasligated with the plasmid pFC107 (Example 9) previously digested by theNruI and SalI restriction enzymes in order to give the plasmid pFC110.

Plasmid pFC110 was linearized by NotI, then transfected in primarychicken embryo cells infected with the canarypox virus (ALVAC strain)according to the method of example 10. A representative plaquecorresponding to in vitro recombination between the donor plasmid pFC110and the genome of the ALVAC canarypox virus was selected on the basis ofa hybridization with a radioactively labelled probe specific to thenucleotide sequence of the envelope glycoprotein E and was thenamplified. The recombinant virus stock obtained was then designatedvCP1714, ALVAC WNV E.

Example 13 Construction of the Recombinant Virus vCP1715

Plasmid pFC102 (Example 4) was digested by the SalI and PmlI restrictionenzymes in order to isolate, following agarose gel electrophoresis, anapproximately 434 by PmIl-SalI restriction fragment. This fragment wasligated with the plasmid pFC107 (Example 9) previously digested by theNruI and SalI restriction enzymes to give the plasmid pFC111.

Plasmid pFC111 was linearized by NotI, then transfected in primarychicken embryo cells infected with the canarypox virus (ALVAC strain)according to the method of Example 10. A representative plaquecorresponding to in vitro recombination between the donor plasmid pFC111and the genome of the ALVAC canarypox virus was selected on the basis ofhybridization with a radioactively labelled probe specific to thenucleotide sequence of the membrane M glycoprotein and was thenamplified. The recombinant virus stock obtained was designated vCP1715,ALVAC WNV M.

Example 14 Construction of the Recombinant Virus vCP1716

Plasmid pFC101 (Example 3) is digested by the SalI and PmlI restrictionenzymes in order to isolate, following agarose gel electrophoresis, anapproximately 484 by PmlI-SalI restriction fragment. This fragment isligated with the plasmid pFC107 (Example 9) previously digested by theNruI and SalI restriction enzymes to give the plasmid pFC112.

Plasmid pFC112 was linearized by NotI and then transfected in primarychicken embryo cells infected with the canarypox virus (ALVAC strain)according to the method of Example 10. A representative plaquecorresponding to in vitro recombination between the donor plasmid pFC112and the genome of the ALVAC canarypox virus was selected on the basis ofa hybridization with a radioactively labelled probe specific to thenucleotide sequence of the pre-membrane prM glycoprotein and was thenamplified. The recombinant virus stock obtained was designated vCP1716,ALVAC WNV prM.

Example 15 Construction of Donor Plasmid for Insertion into C6 Site ofCanarypox Virus (ALVAC)

FIG. 4 of WO01/05934 (see also Audonnet et al., allowed U.S. applicationSer. No. 09/617,594, filed Jul. 14, 2000, now U.S. Pat. No. 6,541,458issued Apr. 1, 2003) shows the sequence of a 3700 by genomic DNAfragment of the canarypox virus. Analysis of this sequence revealed anopen reading frame (ORF) called C6, which starts at position 377 andends at position 2254. The construction of an insertion plasmid leadingto the deletion of the ORF C6 and its replacement by a multiple cloningsite flanked by transcription and translation stop signals wasimplemented in the following way.

A PCR reaction was performed on the basis of the matrix constituted bythe genomic DNA of the canarypox virus and with the followingoligonucleotides:

C6A1 (42 mer) (SEQ ID NO: 20):5′ATCATCGAGCTCGCGGCCGCCTATCAAAAGTCTTAATGAGTT 3′ and C6B1 (73 mer) (SEQID NO: 21): 5′GAATTCCTCGAGCTGCAGCCCGGGTTTTTATAGCTAATTAGTCATTTTTTCGTAAGTAAGTATTTTTATTTAA 3′to isolate a 432 by PCR fragment (fragment D).

A PCR reaction was performed on the basis of the matrix constituted bythe genomic DNA of the canarypox virus and with the followingoligonucleotides:

C6C1 (72 mer) (SEQ ID NO: 22):5′CCCGGGCTGCAGCTCGAGGAATTCTTTTTATTGATTAACTAGTCAAATGAGTATATATAATTGAAAAAGTAA 3′ and C6D1 (45 mer) (SEQ ID NO: 23):5′GATGATGGTACCTTCATAAATACAAGTTTGATTAAACTTAAGTTG 3′to isolate a 1210 by PCR fragment (fragment E).

Fragments D and E were hybridized together to serve as a matrix for aPCR reaction performed with the oligonucleotides C6A1 (SEQ ID NO: 20)and C6D1 (SEQ ID NO: 23) to generate a 1630 by PCR fragment. Thisfragment was digested by the SacI and KpnI restriction enzymes toisolate, after agarose gel electrophoresis, a 1613 by SacI-KpnIfragment. This fragment was ligated with the pBlueScript® II SK+ vector(Stratagene, La Jolla, Calif., USA, Cat #212205) previously digested bythe SacI and KpnI restriction enzymes to give the plasmid pC6L. Thesequence of this plasmid was verified by sequencing. The plasmidcontains 370 by of sequences upstream of ORF C6L (C6 left flanking arm),an early transcription stop vaccinia signal, stop codons in the sixreading frames, a multiple cloning site containing the SmaI, PstI, XhoIand EcoRI restriction sites and finally 1156 by of sequences downstreamof the ORF C6L (C6 right flanking arm).

Plasmid pMPIVC (Schmitt J. F. C. et al., J. Virol., 1988, 62, 1889-1897,Saiki R. K. et al., Science, 1988, 239, 487-491) was used as the matrixfor amplifying the complete sequence of the I3L vaccine promoter withthe following oligonucleotides:

FC112 (33 mer) (SEQ ID NO: 24): 5′AAACCCGGGCGGTGGTTTGCGATTCCGAAATCT 3′and FC113 (43 mer) (SEQ ID NO: 25):5′AAAAGAATTCGGATCCGATTAAACCTAAATAATTGTACTTTGT 3′to amplify a 151 by PCR fragment. This fragment was digested by the SmaIand EcoRI restriction enzymes in order to isolate, following agarose gelelectrophoresis, an approximately 136 by SmaI-EcoRI restrictionfragment. This fragment was then ligated with plasmid pC6L previouslydigested by SmaI and EcoRI to give the plasmid pFC113.

Example 16 Construction of Recombinant Viruses vCP1717 and vCP1718

A PCR reaction was performed using the plasmid pFC106 (Example 8) as thematrix and the following oligonucleotides:

FC114 (33 mer) (SEQ ID NO: 26): 5′TTTCACGTGATGTATAATGCTGATATGATTGAC 3′and FC115 (42 mer) (SEQ ID NO: 27):5′TTTTGGATCCGCGGCCGCTTAACGTTTTCCCGAGGCGAAGTC 3′to amplify an approximately 2973 by PCR fragment. This fragment wasdigested with the PmlI and BamHI restriction enzymes to isolate,following agarose gel electrophoresis, the approximately 2958 byPmlI-BamHI restriction fragment (fragment F). Plasmid pFC113 (example15) was digested by the PmlI and BamHI restriction enzymes to isolate,following agarose gel electrophoresis, the approximately 4500 byPmlI-BamHI restriction fragment (fragment G). Fragments F and G werethen ligated together to give the plasmid pFC114.

Plasmid pFC114 was linearized by NotI, then transfected in primarychicken embryo cells infected with canarypox virus vCP1713 (Example 11)according to the previously described calcium phosphate precipitationmethod (Panicali et Paoletti Proc. Nat. Acad. Sci. 1982, 79, 4927-4931;Piccini et al. In Methods in Enzymology, 1987, 153, 545-563, publishersWu R. and Grossman L. Academic Press). Positive plaques were selected onthe basis of hybridization with a radioactively labelled probe specificto the nucleotide sequence of envelope glycoprotein E NS2A-NS2B. Foursuccessive selection/purification cycles were performed until a purepopulation was isolated. A representative plaque corresponding to invitro recombination between the donor plasmid pFC114 and the genome ofthe ALVAC canarypox virus was then amplified and the recombinant virusstock obtained was designated vCP1717, ALVAC C5 H6p WNV prM-M-E/C6 I3LpWNV NS2A-NS2B-NS3.

The NotI-linearized pFC114 plasmid was also used for transfectingprimary chicken embryo cells infected with the vCP1712 canarypox virus(Example 10) using the procedure described herein. The thus obtainedrecombinant virus stock was designated vCP1718, ALVAC C5 H6p WNVprM-M-E/C6 I3Lp WNV NS2A-NS2B-NS3.

Example 17 Construction of Plasmid pFC115

The complementary DNA (cDNA) of the West Nile fever virus NY99 wassynthesized with Gene Amp RNA PCR Kit (Cat # N 808 0017, Perkin-Elmer,Norwalk, Conn. 06859, USA) using the conditions provided by thesupplier.

A reverse transcriptase polymerase chain reaction (RT-PCR reaction) wascarried out with 50 μl of viral RNA suspension of the West Nile fevervirus NY99 (Example 2) and with the following oligonucleotides:

FC116 (39 mer) (SEQ ID NO: 28) 5′TTTTTTGATATCATGACCGGAATTGCAGTCATGATTGGC3′ and FC106 (33 mer). (SEQ ID NO: 8)

This pair of oligonucleotides makes it possible to incorporate an EcoRVrestriction site, a XbaI restriction site, an initiator code at 5′ and astop code at 3′ of the insert.

Synthesis of the first cDNA strand takes place by elongation of theoligonucleotide FC106, following its hybridization with the RNA matrix.

The synthesis conditions of the first cDNA strand were a temperature of42° C. for 15 min, then 99° C. for 5 min and finally 4° C. for 5 min.The conditions of the PCR reaction in the presence of the pair ofoligonucleotides FC106 and FC116 were a temperature of 95° C. for 2 min,then 35 cycles (95° C. for 1 min, 62° C. for 1 min and then 72° C. for 2min) and finally 72° C. for 7 min to produce a 2079 by fragment.

This fragment was digested by EcoRV and then XbaI to isolate, followingagarose gel electrophoresis, the approximately 2061 by EcoRV-XbaIfragment.

This fragment was ligated with the pVR1012 expression plasmid previouslydigested by XbaI and EcoRV to give the plasmid pFC115 (6956 bp). Underthe control of the early human cytomegalovirus promoter or hCMV-IE(human Cytomegalovirus Immediate Early), this plasmid contains an insertencoding the polyprotein prM-M-E.

Example 18 Construction of the Recombinant Viruses vCP2017-H

A PCR reaction was carried out using the plasmid pFC115 (Example 17) asthe matrix and the following oligonucleotides:

FC117 (36 mer) (SEQ ID NO: 29): 5′TTTTCGCGAATGACCGGAATTGCAGTCATGATTGGC3′ and FC111 (39 mer) (SEQ ID NO: 19)to amplify an approximately 2082 by PCR fragment. This fragment wasdigested by NruI and SalI restriction enzymes to isolate, after agarosegel electrophoresis, an approximately 2071 by NrI-SalI restrictionfragment. This fragment was then ligated with plasmid pFC107 (Example 9)previously digested by the NruI and SalI restriction enzymes to give theplasmid pFC116.

Plasmid pFC116 was linearized by NotI and then transfected in primarychicken embryo cells infected with canarypox virus (ALVAC strain) usingthe procedure of Example 10. A representative plaque corresponding to invitro recombination between the donor plasmid pFC116 and the genome ofthe ALVAC canarypox virus was selected on the basis of a hybridizationwith a radioactively labelled probe specific to the nucleotide sequenceof the envelope glycoprotein E and was then amplified. The recombinantvirus stock obtained was designed vCP2017-H.

Example 18.1 Construction of a C5 H6p WNV prM-M-E Donor Plasmid(pDS-2946-1-1) for the Generation of ALVAC WNV (vCP2017) or ALVAC-2 WNV(vCP2018)

The construction scheme is illustrated in FIG. 1.

A pTriEx-WNV vector containing the 3′-end of the West Nile Virus capsidgene, and the prM/M and E genes with a poly-His tag at the end of the Eprotein from a NY99-related isolate, was obtained from CornellUniversity, and ALVAC (containing WNV prM-M-E in C5 locus) isillustrated in FIG. 1. The WNV E gene contains an internal T5NTsequence, which is known to result in premature transcriptionaltermination in pox-based recombinants (Yuen and Moss, Proc. Natl. Acad.Sci. USA 84:6417-6421, 1987). In order to mutate the T5NT sequence, the1.4 kb Cla I-Xho 13′-WNV fragment from pTriEx-WNV was inserted intopUC-4K, generating clone pDS-2889-1, pUC 3′ WNV. Site-directedmutagenesis was performed using the Amersham QuikChange kit and primers7598.SL (SEQ ID NO: 38) and 7599.SL (SEQ ID NO: 39).

An ApaL I site was introduced for screening purposes. Clone pDS-2897-5-1(pUC 3′-WNV′T5NT) was confirmed as correct by sequence analysis.

In order to remove the poly-His tag and introduce a translation stop andterminal T5NT, the 1.4 kb 3′ fragment from pDS-2897-5-1 was PCRamplified using primers 7617.SL (SEQ ID NO: 42) and 7601.SL (SEQ ID NO:43).

Primer 7601.SL introduces a stop codon, T5NT transcription terminationsignal, and an Xba I site for cloning. The resultant fragment wasinserted into pCR2.1 and clone pDS-2918-1 (pCR2.1 3′-WNV-T5NT+stop) wasconfirmed as correct by sequence analysis.

The 0.7 kb EcoR V-Cla I 5′-end of the WNV gene cassette was PCRamplified using primers 7600.SL (SEQ ID NO: 47) and 7616.SL (SEQ ID NO:48) and the fragment inserted into pCR2.1 to generate plasmidpDS-2905-2-1

The sequence of the insert was confirmed.

Primer 7600.SL contains the 3′-end of the H6 promoter from the NruI siteand primer 7616.SL spans a Cla I site in the WNV E gene. The EcoR V-ClaI 5′-fragment from pDS-2905-2-1 and the Cla I-Xba I 3′ fragment frompDS-2918-1 were inserted into the ALVAC C5 donor plasmid pNVQH6C5LSP-18(pC5 H6p donor plasmid) that had been digested with EcoR V and Xba I.Clone pDS-2946-1-1 was confirmed by restriction enzyme analysis and theH6p WNV prM-M-E insert was confirmed by sequence analysis. The sequenceof the C5-H6p WNV prM-M-E-C5 gene cassette is illustrated in FIG. 2, andthe full sequence is illustrated in FIG. 9. From the sequences depictedin FIG. 2 and FIG. 9, one of skill in the art may clone these sequencesand easily generate the plasmids herein without following this exactprocedure.

Alternatively, plasmids with a truncated H6p and/or truncated WNV capsidleader sequence may be useful (see FIG. 10 and FIG. 11).

Example 18.2 Generation and Characterization of ALVAC WNV, (vCP2017) andALVAC-2 WNV (vCP2018)

ALVAC-2 is a canarypox virus containing an E3LK3L gene cassette insertedat the unique C6 locus of ALVAC and is described in U.S. Pat. No.5,756,103. To generate ALVAC- or ALVAC-2-based WNV recombinants, primarychick embryo firbroblast cells (CEFs) were transfected with NotI-linearized pDS-2946-1-1 plasmid DNA (pC5 H6p WNV prM-M-E) mixed withFuGENE-6 transfection reagent (Roche), then infected with ALVAC orALVAC-2 as rescue virus at an MOI of 10. After 24-48 hours, recombinantplaques were lifted onto nylon membrane and hybridized with aWNV-specific DNA probe which was labelled with horseradish peroxidaseaccording to the manufacturer's protocol (Amersham Cat# RPM3001).Following 2-4 sequential rounds of plaque purification, single plaqueswere amplified to produce stocks of vCP2017 and vCP2018. Recombinantviruses were characterized by restriction enzyme and Southern blotanalyses. The C5-H6p WNV-05 locus was PCR-amplified and the completesequence confirmed. Expression of the WNV proteins was confirmed byimmunoplaque and immunoblot analyses. Both of these recombinants containtwo copies of the H6p WNV prM-M-E genes.

Example 19 Production of Recombinant Vaccines

For the preparation of equine vaccines, the recombinant canarypoxvCP1712 virus (Example 10) is adjuvanted with carbomer solutions, namelyCarbopol™974P manufactured by BF Goodrich, Ohio, USA (molecular weightabout 3,000,000).

A 1.5% Carbopol™974P stock solution was initially prepared in distilledwater containing 1 g/l of sodium chloride. This stock solution was thenused for the preparation of a 4 mg/ml Carbopol™974P solution inphysiological salt solution. The stock solution was mixed with theadequate volume of the physiological salt solution, either in a singlestage or in several successive stages, the pH value being adjusted ineach stage with a 1N sodium hydroxide solution (or even moreconcentrated) in order to obtain a final pH value of 7.3 to 7.4.

The ready-to-use Carbopol™974P solution obtained in this way was usedfor taking up recombinant, lyophilized viruses or for dilutingconcentrated, recombinant virus stock solutions. For example, to obtaina viral suspension containing 10⁸ pfu/1 ml dose, a viral stock solutionwas diluted so as to obtain a titer of 10^(8.3) pfu/ml, followed bydilution in equal parts with said ready-to-use 4 mg/ml Carbopol™974Psolution.

Recombinant vaccines can also be produced with recombinant canarypoxviruses vCP1713 (Example 11) or vCP1717 (Example 16) or vCP1718 (Example16) or vCP2017 (Example 18.1) or a mixture of three canarypox virusesvCP1714 (Example 12), vCP1715 (Example 13) and vCP1716 (Example 14)according to the procedure described herein.

Example 20 Production of DNA Vaccines for Equines

A DNA solution containing the plasmid pFC104 (Example 6) wasconcentrated by ethanol precipitation in the manner described bySambrook et al (1989). The DNA sediment was taken up by a 0.9% NaClsolution so as to obtain a concentration of 1 mg/ml. A 0.75 mMDMRIE-DOPE solution is prepared by taking up a DMRIE-DOPE lyophilizateby a suitable sterile H₂O volume.

The formation of plasmid-lipid DNA complexes was brought about bydiluting in equal parts the 0.75 mM DMRIE-DOPE solution (1:1) with the 1mg/ml DNA solution in 0.9% NaCl. The DNA solution was progressivelyintroduced with the aid of a 26G crimped needle along the wall of theflask containing the cationic lipid solution so as to prevent theformation of foam. Gentle stirring takes place as soon as the twosolutions mixed. Finally a composition comprising 0.375 mM of DMRIE-DOPEand 500 μg/ml plasmid was obtained.

It is desirable for all the solutions used to be at ambient temperaturefor all the operations described herein. DNA/DMRIE-DOPE complexing takesplace at ambient temperature for 30 minutes before immunizing theanimals.

DNA vaccines can also be produced with DNA solutions containing plasmidspFC104 (Example 6) and pFC106 (Example 8) or containing plasmids pFC105(Example 7) and pFC106, plasmids pFC115 (Example 17) and pFC106, orcontaining plasmid pFC101, pFC102 and pFC103 (Examples 3 to 5), orcontaining plasmid pFC105 or pFC115 according to the procedure describedin the present Example.

Example 21 In Vitro Expression Tests

The expression of WN proteins was tested for each construction byconventional indirect immunofluorescence and Western Blot methods.

These tests were carried out on 96 well plates containing CHO cellscultured in monolayers and transfected by plasmids or containing CEFcells cultured in monolayers and infected by recombinant viruses.

The WN proteins were detected by the use of infected chicken or horsesera and of labelled anti-sera.

The size of the fragments obtained after migration on agarose gel wascompared with those expected.

Example 22 Effectiveness on Animals

The recombinant vaccines and plasmid vaccines were injected twice atapproximately two week intervals into approximately seven day old,unvaccinated SPF chickens by the intramuscular route and in a volume ofapproximately 0.1 ml. An unvaccinated control group was included in thestudy.

The chickens were challenged by subcutaneous administration into theneck of 10^(3.5)TCID₅₀ of pathogenic WN virus.

Viremia, antibody response and mortality were observed. Autopsies werecarried out to observe lesions.

Example 23 Titrating Anti-WNV Neutralizing Antibodies

Dilution series were produced for each serum at a rate of 3 in DMEMmedium to which was added 10% fetal calf serum in 96 well plates of thecellular culture type. To 0.05 ml of diluted serum was added 0.05 ml ofculture medium containing approximately 100 CCIP₅₀/ml of WNV. Thismixture was incubated for 2 hours at 37° C. in an oven in an atmospherecontaining 5% CO2.

0.15 ml of a suspension of VERO cells containing approximately 100,000cells/ml was then added to each mixture. The cytopathic effect (CPE) wasobserved by phase contrast microscopy after 4 to 5 days culturing at 37°C. in an atmosphere containing 5% CO₂. The neutralizing titers of eachserum were calculated using the Kärber method. The titers were given inthe form of the largest dilution inhibiting the cytopathic effect for50% of the wells. The titers were expressed in log 10 VN50. Each serumwas titrated at least twice and preferably four times.

Example 24 Test on Horses of vCP2017

Recombinant vaccines containing vCP2017 (Example 18.1) formulatedextemporaneously with 1 ml of Carbopol© 974P adjuvant (4 mg/ml) wereinjected twice at 35 day intervals into horses aged more than threemonths and which had not been previously vaccinated, using theintramuscular route and a volume of approximately 1 ml. Three groups ofanimals were vaccinated, with doses of 10^(5.8)CCID₅₀ (i.e. 10^(5.64)pfu) for group 1, 10^(6.8)CCID₅₀ (i.e. 10^(6.64) pfu) for group 2 and10^(7.8)CCID₅₀ (i.e. 10^(7.64) pfu) for group 3. An unvaccinated controlgroup was included in the study.

The neutralizing antibody titers were determined as indicated in Example23 The titers were expressed in log 10 VN50.

Group Titers at day 0 Titers at day 35 Titers at day 49 1 <0.6 <0.782.66 2 <0.6 1.14 2.58 3 <0.6 1.16 2.26 Control <0.6 <0.6 <0.6

Example 25 Protection after Challenge in Horses

Twenty horses (mares), 3-11 years old, were randomly allocated into twogroups of 10 horses each. 1 ml of vaccine containing the recombinantvCP2017 10^(6.3) CCID₅₀ (example 18.1) was formulated extemporaneouslywith 1 ml of Carbopol© 974P adjuvant at 4 mg/ml. Horses of group 1 wereinjected twice at 35 day intervals using the intramuscular route and avolume of approximately 1 ml containing 10^(6.0)CCID₅₀ (i.e. 10^(5.84)pfu). One of the vaccinated horses had to be removed from the studyprior to challenge due to recurrent colic.

Horses of group 2 remained unvaccinated and served as controls.

Horses from both groups were challenged on day 49 with WNV via the bitesfrom WNV-infected Aedes albopictus mosquitoes. The Aedes albopictusmosquitoes were infected intrathoracically with WNV NY99 eight daysprior to the challenge. Each mosquito received approximately 150 pfu. Atchallenge, a round carton capped with a fine nylon mesh (containingapproximately 20 WNV-infected Aedes albopictus mosquitoes) was held(mesh side down) over a clipped area of the horse skin for 5 to 8minutes.

The neutralizing antibody titers were determined as indicated in example23. The titers were expressed in log 10 VN50.

Group D 0 D 35 D 42 D 49 D 63 Vaccinated <0.84 <0.93 2.42 2.78 3.36Control <0.72 <0.75 <0.78 <0.78 3.43

None of the 9 vaccinated horses developed detectable WNV viremia while 8of 10 control horses developed detectable WNV viremia.

Example 26 Test on Cats of vCP2017

41 cats, 14-20 weeks old, were randomly allocated into four groups.

Vaccines containing the recombinant vCP2017 (example 18.1) in 1 ml ofsterile water per dose were injected twice at 28 day intervals via thesubcutaneous route. Three groups of animals were vaccinated, with dosesof 10^(7.9)CCID₅₀ (i.e. 10^(7.74) pfu) for group 1 (8 cats),10^(6.4)CCID₅₀ (i.e. 10^(6.24) pfu) for group 2 (14 cats) and10^(5.9)CCID₅₀ (i.e. 10^(5.74) pfu) for group 3 (8 cats).

An unvaccinated control group was included in the study. The 11 cats ofthis group received placebo injections (1.0 ml of Phosphate Bufferedsaline (PBS) subcutaneously, twice 4 weeks apart).

The neutralizing antibody titers in serum were determined according toexample 23. The titers were expressed in log 10 VN50.

Group D0 D28 D42 Control <1.01 <1.01 <1.03 1 <0.9 <1.08 2.26 2 <1.08<0.99 2.16 3 <0.95 <0.97 1.36

The neutralizing antibody titers in serum were determined with PRNTmethod (plaque reduction neutralization test; see Bunning M. L. et al.,Emerging infectious diseases, 8(4), 380-386, 2002). The titers wereexpressed as a dilution starting from 1:5. The mean PRNT results at day42 at 90%, 80% and 50% reduction by group:

Titers at day 42 Titers at day 42 (reduction (reduction of Titers at day42 Group of 90%) 80%) (reduction of 50%) Control 5.00 5.00 5.00 1 16.8829.38 55.63 2 15.36 11.92 45.36 3 5.00 5.00 6.25

Example 27 Protection after Challenge in Cats

The cats of the groups 2, 3 and control of the example 26 werechallenged, 4 months after the second injection (example 26), with WNVvia the bites of WNV-infected Aedes albopictus mosquitoes. The Aedesalbopictus mosquitoes were infected intrathoracically with WNV NY99 8-10days prior to the challenge. Each mosquito received approximately 150pfu. At challenge, a round carton capped with a fine nylon mesh(containing approximately 5-15 WNV-infected Aedes albopictus mosquitoes)was held (mesh side down) over a clipped area of the cat skin for 5 to10 minutes. The feeding of mosquitoes was confirmed by visualization ofengorgement.

A representative sample of engorged, infected mosquitoes was titratedfor WNV in order to determine the infection rate of mosquitoes. About 3representative engorged mosquitoes from each cat were titrated for WNV.The results are 8.4 log pfu/mosquito for control group cats, 8.4 logpfu/mosquito for group 2 cats and 8.3 log pfu/mosquito for group 3 cats.

The neutralizing antibody titers and post-challenge WNV viremia weredetermined. The titers were calculated with PRNT method (plaquereduction neutralization test) and expressed in dilution starting at1:5.

The mean PRNT results at 90% reduction by group and by post-challengeday:

Group D0 D7 D14 Control 5.00 5.00 20 2 7.14 89.64 148.57 3 5.00 47.5047.50

The mean PRNT results at 80% reduction by group and by post-challengeday:

Group D0 D7 D14 Control 5.00 5.45 21.82 2 9.29 101.43 214.29 3 5.6353.75 125.00

Incidence of WN virus isolation (number of cats having a positive WNvirus isolation) by group and by post-challenge day:

Day day day day day day day days day Group 0 1 2 3 4 5 6 7-10 14 Control0 0 3 5 7 7 5 0 0 2 0 0 0 0 0 0 0 0 0 3 0 0 1 0 0 0 0 0 0

None of the 14 vaccinated cats of group 2 developed a detectable WNVviremia, only one of the 8 vaccinated cats of group 3 developed adetectable WNV viremia while 9 of 11 control cats developed a detectableWNV viremia.

Example 28 Construction of the Recombinant Viruses vFP2000

As illustrative of one embodiment of the invention a specific fowlpoxrecombinant construct is described in this Example.

A PCR reaction was performed on the basis of the matrix constituted bygenomic DNA of a fowlpox virus (DIFTOSEC CT©, strain marketed by MERIAL)and with the following oligonucleotides:

F8FCA1 (42 mer) (SEQ ID NO: 30):5′ATCATCGAGCTCGACCCTTTACAAGAATAAAAGAAGAAACAA 3′ and F8FCB1 (73 mer) (SEQID NO: 31): 5′CTCGAGCTGCAGGAATTCCCCGGGTTTTTATTAGCTAATTAGCAATATAGATTCAATATGATAATTACTCTAA 3′in order to isolate a 1483 by PCR fragment (fragment H).

A PCR reaction was carried out on the basis of the matrix constituted bygenomic DNA of the fowlpox virus and with the followingoligonucleotides:

F8FCC1 (72 mer) (SEQ ID NO: 32):5′CCCGGGGAATTCCTGCAGCTCGAGTTTTTATTGACTAGTTAATCATAAGATAAATAATATACAGCATTGTAA 3′ and F8FCD1 (45 mer) (SEQ ID NO: 33):5′GATGATGGTACCGGGTAATGGCTTTTGTTTATAACCACGTTTGTC 3′in order to isolate a 1433 by PCR fragment (fragment I).

Fragments H and I were hybridized together in order to serve as a matrixfor a PCR reaction performed with the oligonucleotides F8FCA1 (SEQ IDNO: 30) and F8FCD1 (SEQ ID NO: 33) in order to generate a 2892 by PCRfragment. This fragment was digested by the restriction enzymes SacI andKpnI in order to isolate, following agarose gel electrophoresis, a 2875by SacI-KpnI fragment. This fragment was ligated with the pBlueScript®II SK+ vector (Stratagene, La Jolla, USA, Cat #212205), previouslydigested by the restriction enzymes SacI and KpnI, in order to give theplasmid pF8L. The sequence of this plasmid was verified by sequencing.This plasmid contains 1424 by of sequences upstream of ORF F8 (leftflanking arm F8), an early transcription stop vaccine signal, stopcodons in 6 reading frames, a multiple cloning site containingrestriction sites SmaI, PstI, XhoI and EcoRI and finally 1376 by ofsequences located downstream of ORF F8 (right flanking arm F8).

The plasmid pMP528HRH (Perkus M. et al. J. Virol. 1989, 63, 3829-3836)was used as the matrix for amplifying the complete sequence of thevaccine promoter H6 (GenBank accession no. M28351) with the followingoligonucleotides:

FC125 (95 mer) (SEQ ID NO: 34)5′AAACCCGGGTTAATTAATTAGTCATCAGGCAGGGCGAAACGAGACTATCTGCTCGTTAATTAATTAGAGCTTCTTTATTCTATACTTAAAAAGTG 3′ and FC126 (43 mer)(SEQ ID NO: 35) 5′AAAACTGCAGGTCGACTACGATACAAACTTAACGGATATCGCG 3′in order to amplify a 211 by PCR fragment. This fragment was digested byrestriction enzymes SmaI and PstI in order to isolate, following agarosegel electrophoresis, a 200 by SmaI-PstI restriction fragment. Thisfragment was then ligated with the plasmid pF8L, previously digested bySmaI and PstI, in order to give the plasmid pFC121.

A PCR reaction was performed using the plasmid pFC115 (example 17) asthe matrix and the following oligonucleotides:

FC127 (58 mer) (SEQ ID NO: 36)5′ TTTTCGCGATATCCGTTAAGTTTGTATCGTAATGACCGGAATTGCAG TCATGATTGGC 3′ andFC128 (43 mer) (SEQ ID NO: 37)5′ TTTTGTCGACTCTAGATAAAAATTAAGCGTGCACGTTCACGGA 3′in order to amplify an approximately 2111 by PCR fragment. This fragmentwas digested by restriction enzymes NruI and SalI in order to isolate,following agarose gel electrophoresis, an approximately 2096 byNruI-SalI restriction fragment. This fragment was then ligated withplasmid pFC121 previously digested by restriction enzymes NruI and XhoIin order to give the plasmid pFC122.

Plasmid pFC122 was linearized by PvuI, then transfected in primarychicken embryo cells infected with the fowlpox virus according to thepreviously described calcium phosphate precipitation method (Panicali etPaoletti Proc. Nat. Acad. Sci. 1982, 79, 4927-4931; Piccini et al. InMethods in Enzymology, 1987, 153, 545-563, publishers Wu R. and GrossmanL. Academic Press). Positive plaques were selected on the basis of ahybridization with a radioactively labelled probe specific to thenucleotide sequence of the envelope glycoprotein E. These plaquesunderwent 4 successive selection/purification cycles until a purepopulation was isolated. A representative plaque corresponding to invitro recombination between the donor plasmid pFC122 and the genome ofthe fowlpox virus was then amplified and the recombinant virus stockobtained was designated vFP2000, fowlpox WNV prM-M-E.

Example 28.1 Construction of a pF8 H6p WNV prM-M-E Donor Plasmid(pSL-5513-1-1-1) for the Generation of Fowlpox WNV (vFP2000)

The construction scheme is illustrated in FIG. 3.

There is one commonly used recombination site in the Fowlpox genome,designated as F8. Plasmid pMAW112-2/F8 AIV HA has been described inpatent application xxx and was used as the source of the Fowlpox F8arms. The 1.7 kb AIV HA insert in pMAW112-2/F8 AIV HA was deleted bydigestion with Nru I and Hind III, to be replaced by oligonucleotides7737.SL (SEQ ID NO:52) and 7738.SL (SEQ ID NO:53) encoding the 3′-end ofthe H6 promoter and a Sma I/Xma I site. The correct insertion of theoligos was confirmed in plasmid pF8 H6p MCS, pSL-5440-5-1. This plasmidcontains ˜1.4 kb of the upstream F8 flanking sequence, designated as F8Rand ˜1.4 kb of the downstream flanking sequence of F8, designated asF8L, as well as the H6 promoter and a multiple cloning site of Xma I,Hind III, BamH I and Xho I.

Plasmid pDS-2946-1-1 is an ALVAC donor plasmid that contains the H6promoter and West Nile Virus prM-M-E genes, between the C5 arms and isdescribed in Example 18.1 and FIG. 1. pDS-2946-1-1 was digested with BglII and Xba I and the 2.8 kb C5R-H6p WNV fragment was inserted intopT7-7, between Bgl II and Xba I, generating plasmid pT7C5R-WNV,pSL-5501-2. Plasmid pSL-5501-2 was digested with Nru I and SalI and the2.1 kb fragment inserted into pSL-5440-5-1 that had been digested withNru I and Xho. The resultant pF8 H6p WNV prM-M-E plasmid,pSL-5513-1-1-1, was confirmed by restriction enzyme digestion andnucleotide sequence analysis. The sequence of the F8-H6p WNV prM-M-E-F8gene cassette from pSL-5513-1-1-1 is shown in FIG. 4.

Example 28.2 Generation and Characterization of a Fowlpox RecombinantExpressing West Nile Virus prM-M-E, vFP2000

Primary CEFs were transfected with Not I-linearized pSL-5513-1-1-1plasmid DNA (15 ug) using Fugene reagent (Roche) by the method suggestedby the supplier. The transfected cells were subsequently infected withfowlpox as rescue virus at MOI of 10 and after ˜24 h, thetransfected-infected cells were harvested, sonicated and used forrecombinant virus screening. Recombinant plaques were screened based onthe plaque lift hybridization method using a WNV-specific probe, whichwas directly labelled with horseradish peroxidase according to themanufacturer's protocol (Amersham). After five sequential rounds ofplaque purification, the recombinants designated as vFP2000.2.1.1.1.1and vFP2000.3.2.1.2.1 were generated and confirmed by hybridization as100% positive for the WNV insert and 100% negative for the F8 ORF.Agarose punches were selected from the fifth round of plaquepurification, and expanded to obtain stocks of vFP2000. Recombinantviruses were characterized by restriction enzyme and Southern blotanalyses. The F8-H6p WNV-F8 locus was PCR-amplified and the completesequence confirmed. Expression of the WNV proteins was confirmed byimmunoplaque and immunoblot analyses. The fowlpox recombinants containone copy of the H6p WNV prM-M-E gene cassette.

Example 29 Test on Geese of vFP2000

Chinese geese, one-weeks old, were randomly allocated into four groups.

Vaccine was prepared by mixing extemporaneously 1 ml of the recombinantvFP2000 (Example 28) at 10^(6.3) CCID₅₀ with 1 ml of Carbopol© 974Padjuvant at 4 mg/ml. One group of 5 birds was vaccinated with 0.2 ml bythe intramuscular route twice at 13 day interval (called vFP2000 group).

Two unvaccinated control groups were included in the study. One controlgroup of 7 geese was not vaccinated and was not challenged (called shamcontrol group). One control group of 8 geese was not vaccinated but waschallenged (called challenged control group). The geese of these groupsreceived by intramuscular route placebo injections (0.2 ml of aCarbopol© 974P solution at 2 mg/ml) twice 13 days apart.

Geese from the vFP2000 group and from the control group were challengedon day 26 with 0.2 ml containing about 10^(3.5) CCID₅₀ of WNV bysubcutaneous route.

The morbidity and post-challenge WNV viremia were observed. The virustiter was calculated and expressed as TCID₅₀/0.1 ml virus titers.

Group D o D 1 D 2 D 3 D 4 D 5 D 7 D 10 Chal- <0.3 4.2 4.5 3.3 3.9 0.7<0.3 <0.3 lenged control vFP2000 <0.3 0.5 0.6 0.5 0.8 <0.3 <0.3 <0.3Sham <0.3 <0.3 <0.3 <0.3 <0.3 <0.3 <0.3 <0.3 control

The viremia was expressed as the percentage of WNV-excreting animals foreach group.

Group D 0 D 1 D 2 D 3 D 4 D 5 D 7 D 10 Chal- 0% 87.5% 100% 100% 100%62.5% 0% 0% lenged control vFP2000 0%   20%  40%  20%  40%   0% 0% 0%Sham 0%   0%  0%  0%  0%   0% 0% 0% control

None of the five vFP2000 vaccinated geese developed detectable morbiditywhile 4 of 8 challenged control geese developed detectable morbidity.None of the sham control geese developed detectable morbidity.

Example 30 Immunoblot Analysis of the Expression of WNV Proteins fromALVAC WNV (vCP2017), ALVAC-2 WNV (vCP2018) and Fowlpox WNV (vFP2000)Recombinants

ALVAC recombinants vCP2017 and vCP2018 each contain two copies of theH6p WNV prM-M-E gene cassette located at the C5 loci, while vFP2000contains a single copy of H6p WNV prM-M-E at the F8 locus. CEFs wereinfected with vCP2017, vCP2018 or vFP2000 at an MOI of 10 and grown inserum-free DMEM medium for 24 and 48 hours at 37° C., 5% CO₂. Theculture medium was collected and the cells were resuspended in PBS, thencentrifuged and the PBS discarded. Cell lysate samples were prepared byresuspending the pellets in water, adding 5×SDS-PAGE sample buffer(without β-mercaptoethanol), and boiling for 5 minutes. Supernatantsamples were prepared by adding 5×SDS-PAGE sample buffer (withoutβ-mercaptoethanol), and boiling for 5 minutes. Samples were run on a 12%SDS-PAGE gel and the separated proteins were transferred to nylonmembrane (Millipore Immobilon P). The immunoblot was probed with chickenanti-WNV antisera (Merial Ltd.), then horseradish peroxidase conjugateddonkey-anti-chicken antibody (Jackson Labs) was used as secondaryantibody. The reactive bands were visualized with ChemiluminescenceReagent (Perkin Elmer). As illustrated in FIG. 5, the expression of boththe WNV E and M proteins was higher in the cell fraction from thefowlpox recombinant than from the ALVAC recombinants. The production ofthe WNV E protein was also higher in the supernatant fraction from thefowlpox recombinant than the ALVAC recombinants, but there was no Mprotein secreted.

In order to determine whether this finding was reproducible in non-aviancells, baby hamster kidney (BHK) cells were infected with vCP2017,vCP2018 or vFP2000 at an MOI of 10. After ˜24 h, culture supernatantsand cells were collected, as described above. For SDS-PAGE analysis, 5×sample buffer (without β-mercaptoethanol) was added to the culturesupernatants and the samples boiled for 5 min. The infected cells wereresuspended in PBS, then spun to concentrate them and for SDS-PAGEanalysis, lysis buffer was added, samples boiled for 5 min, then 5×sample buffer was added and the samples re-boiled for 5 min. Sampleswere separated on a 12% SDS-PAGE gel and proteins transferred toMillipore Immobilon P nylon membrane. Chicken anti-WNV antibody (MerialLtd.) was used as primary antibody and horseradish peroxidase-conjugateddonkey-anti-chicken antibody (Jackson Labs) was used as secondaryantibody. Reactive protein bands were visualized with ChemiluminescenceReagent (Perkin Elmer). As illustrated in FIG. 6, the expression of theWNV E protein in the supernatant fraction from the fowlpox recombinantwas higher than the ALVAC recombinants, in mammalian cells. There was noobvious difference in expression levels for the various recombinants inthe cell fraction, however the fowlpox recombinant has only one genecopy compared to two gene copies in the ALVAC vectors.

Example 31 Generation and Characterization of Specific Antisera to WestNile Virus Proteins Expressed by ALVAC WNV (vCP2017), ALVAC-2 WNV(vCP2018) and Fowlpox WNV (vFP2000) Recombinants

The in vitro expression of West Nile Virus M and E proteins from ALVACor fowlpox recombinants in primary CEFs and BHK cells was demonstratedin Example 30. In order to determine whether the recombinants wereimmunogenic and if there was a demonstrable difference in the quality ofthe antisera, mice were immunized with vCP2017, vCP2018 and vFP2000.Balb/c mice (Charles River, Quebec) were immunized IM with 4×10⁷ pfu ofrecombinant virus in 1 mM PBS, at days 0 and 21. Blood from day 35 wasprocessed and used to probe immunoblots. Samples for immunoblot analysisof antisera from ALVAC WNV immunizations were prepared by transienttransfection of Chinese hamster ovary (CHO) cells with pTriEx-WNV DNA,which had been shown to express the WNV M and E genes. CHO cells weretransfected with 10 ug of pTriEx-WNV DNA, using electroporation atsettings 1.5 kV, 25 uF and infinite resistance. Mock samples wereprepared by the electroporation of CHO cells at the same settings,without DNA. After approximately 65 hours, supernatants were harvestedand clarified by spinning at 3000 rpm for 5 min. The plates were washedtwice with PBS, then 500 μl of PBS was added and the cells were scrapedoff. After spinning at 3000 rpm for 5 min, the supernatant was removedand the cells resuspended in 100 μl of SDS-PAGE lysis buffer. Sampleswere suspended in SDS-PAGE loading buffer (minus β-mercaptoethanol) andseparated on a 12% SDS-PAGE gel before electrotransfer to Immobilon Pnylon membrane. The membranes were processed and probed with 1:1000dilution of the mouse anti-WNV antisera. Peroxidase-conjugated goatanti-mouse antisera (Jackson Labs) were used as secondary antibody at adilution of 1:500 and bands were visualized using ChemiluminescenceReagent (Perkin Elmer). Five of five mouse anti-WNV antisera generatedby vCP2017 immunization, reacted specifically with a protein band in thepTriEx-WNV samples, of the expected size for the WNV E protein. All ofthese antisera also reacted with media components in both the pTriEx-WNVand mock samples. Four of five mouse anti-WNV antisera generated byvCP2018 immunization, reacted specifically with a protein band in thepTriEx-WNV samples, of the expected size for the WNV E protein. All ofthe five antisera also reacted with media components in both thepTriEx-WNV and mock samples.

For analysis of anti-WNV antibodies following vFP2000 immunization,vFP2000 pellet and supernatant samples were run on 12% SDS-PAGE gels andtransferred to Immobilon P nylon membrane (Millipore). The membraneswere processed and probed with 1:1000 dilution of the mouse anti-WNVantisera. Peroxidase-conjugated goat anti-mouse antibody (Jackson Labs)was used as secondary antibody at a dilution of 1:500 and bands werevisualized using Chemiluminescence Reagent (Perkin Elmer). Five of fivemouse anti-WNV antisera generated by vFP2000 immunization, recognizedspecific bands in the vFP2000 samples, corresponding to WNV E and Mproteins. Anti-vFP2000 antisera also recognized specific WNV bandsexpressed in vCP2017 and vCP2018.

Example 32 One Dose Efficacy of a Canarypox Vectored West Nile Virus(WNV) Vaccine (vCP2017) Against a WNV-infected Mosquito Challenge inHorses

The efficacy of a single dose of a canarypox vectored West Nile Virusvaccine comprising vCP2017 (described in Example 18.1, above) was testedin horses. After acclimation, the animals were randomly assigned to eachof two treatment groups: 19 horses were divided into Group I (9 horses,vaccinated) and Group II (10 horses, control). The clinician performinglaboratory analyses and clinical observations was blind to the groupassignment.

Each of the 9 horses in Group I was intramuscularly vaccinated in thelateral cervical area on Day 0 with a 1 ml dose of vaccine containing10E06 TCID50 of the recombinant canarypox VCP 2017 (described in Example18.1, above) and 4 mg of Carbopol 974P. The 10 horses of the controlgroup (Group II) remained unvaccinated. Blood samples were collected onDays 0, 7, 14, 21, and 26 before challenge and on Days 33 and 40post-challenge to test the presence of neutralizing antibodies.

Aedes albopictus mosquitoes were infected intrathoracically with WNVNY99, 7-14 days prior to the challenge. A representative sample of thepool of infected mosquitoes was titrated for WNV in order to determinethe infection rate of the mosquitoes and the variability of virus loadper mosquito. Each horse was challenged on Day 26 by the bite of 10-20of the infected Aedes. Mosquitoes were allowed to feed on the horses for5-10 minutes.

After challenge, the mosquitoes were chilled in a refrigerator, sortedand classified as engorged or not. The number of engorged mosquitoes wasrecorded and the engorged mosquitoes were frozen at −75° C. and helduntil the virus assay was performed (WNV post-challenge titration). Themosquitoes were homogenized in 1 ml BA1 and titrated by plaque assay onVero cells. The quantity of West Nile virus present in the engorgedmosquitoes after feeding on the horses ranged between 10E07 to 10E09 pfuper mosquito.

All 19 animals were observed for depression, neurological signs andhyperthermia. Blood samples were collected twice daily on Days 26 to 40for detection of West Nile virus viremia.

Animals were observed for depression and neurological signs (ataxia,head shaking, muscle fasciculation, reluctance to move, anxiety and liptwitching) were recorded as present or absent. Examination also includedrecording of rectal temperatures (° F.). Data collected prior tochallenge were used to establish the baseline for clinical signs.

All sera was tested for the presence of SN antibodies against West NileVirus using the Plaque Endpoints determined at 50%, 80%, and 90%plaque-reduction levels. Titrations started at 1:5 dilution. Titers≦1:5at 50% plaque reduction were considered negative. In this assay, 100 to150 plaque-forming units per well were used.

Blood samples were tested by plaque titration for detection of WNVviremia. Viremia was reported as negative (titer of <5 pfu/ml[log₁₀≧0.7]) or positive (titer of ≧5 pfu/ml [log₁₀≧0.7]).

One out of nine vaccinated horses of Group I developed detectable WestNile virus viremia (11.1%), whereas eight out ten control horses (GroupII) developed detectable viremia (80%) post challenge (see FIGS. 16, 17a-b).

One unvaccinated horse had a single episode of fever (102° F.) postchallenge (see FIG. 18 a-c). There was no incidence of any clinicalsigns after challenge. None of the challenged horses died from exposureto West Nile virus infection. Additionally, there were no incidences ofany clinical signs that were observed for this study in any of thechallenged horses.

Prior to vaccination all horses were seronegative (titer<1:5). On Days14, 21, 26 three vaccinated horses had positive West Nile neutralizingantibody titers (titer> or =1:5 at 80% plaque reduction according to thePlaque reduction neutralization test [PRNT]). All control animalsremained negative. The data is shown in FIGS. 19 a-b.

* * *

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A composition comprising a recombinant poxvirusor DNA plasmid comprising a polynucleotide, wherein the polynucleotideencodes and expresses WVN (West Nile Virus) prM-M-E having the aminoacid sequence as set forth in SEQ ID NO:59.
 2. A composition comprisinga recombinant poxvirus or DNA plasmid comprising a polynucleotide,wherein the polynucleotide encodes and expresses WVN E having the aminoacid sequence corresponding to amino acid positions 186-686 of SEQ IDNO:59.
 3. A composition comprising a recombinant poxvirus or DNA plasmidcomprising a polynucleotide, wherein the polynucleotide encodes andexpresses WVN M-E having the amino acid sequence corresponding to aminoacid positions 111-686 of SEQ ID NO:59.
 4. A composition comprising arecombinant poxvirus or DNA plasmid comprising a polynucleotide, whereinthe polynucleotide encodes and expresses WVN prM-M-E having the aminoacid sequence corresponding to amino acid positions 19-686 of SEQ IDNO:59.
 5. A composition comprising a recombinant poxvirus or DNA plasmidcomprising a polynucleotide, wherein the polynucleotide encodes andexpresses WVN prM-E having the amino acid sequence corresponding toamino acid positions 19-110 and 186-686 of SEQ ID NO:59.
 6. Thecomposition of any one of claims 1 and 2-5, wherein the recombinantpoxvirus is a canarypox or fowlpox virus.
 7. The composition of claim 6,wherein the canarypox virus is ALVAC and the fowlpox virus is TROVAC. 8.The composition of any one of claims 1 and 2-5, wherein the DNA plasmidis pVR1020 or pVR1012.
 9. The composition of any one of claims 1 and 2-5further comprising an adjuvant.
 10. The composition of claim 9, whereinthe adjuvant is a carbomer.
 11. The composition of any one of claims 1and 2-5 further comprising an antigen or immunogen or epitope thereof ofa pathogen other than WNV of the animal, or a vector that contains andexpresses in vivo in the animal a nucleic acid molecule encoding theantigen, immunogen or epitope thereof, or an inactivated or attenuatedpathogen other than WNV of the animal.
 12. A method for inducing aprotective immune response against WNV in an animal comprisingadministering to the animal the composition of any one of claims 1 and2-5.
 13. The method of claim 12 additionally comprising an adjuvant. 14.The method of claim 13, wherein the adjuvant comprises a carbomer.
 15. Amethod for inducing a protective immune response against WNV and asecond pathogen in an animal comprising administering to the animal thecomposition of claim
 11. 16. A method for inducing a protective immuneresponse against WNV in an animal comprising administering to the animal(a) the composition of any one of claims 1 and 2-5, and (b) aninactivated WNV vaccine, an attenuated WNV vaccine, or a subunit vaccinecomprising WNV isolated antigen or immunogen, wherein (a) isadministered prior to (b) in a prime-boost regimen, or (b) isadministered prior to (a) in a prime-boost regimen, or (a) and (b) areadministered together, either sequentially or in admixture.
 17. Themethod of claim 12, wherein the animal is equine, avian, feline, orcanine.